Protecting Newcastle Disease Vaccine with Intrinsically Disordered Proteins Igem 2019

Protecting Newcastle Disease Vaccine with Intrinsically Disordered Proteins iGEM 2019

Andrew Zarzar1, Aren Pageler1, Ikenna Anigbogu1, James Hahn1, Jiasui Xu1, Jonah Pierce1, Liana Beld2, Manpreet Kaur1, Marcella Mirabelli2, Marcus-Charles Strauss3, Martina Pedersen2, Melody Azimi2, Nicolasa Cardenas1, Rita Ousterhout1, Shayan Vahdani1, Varuna Dharasker1,2

University of California, Santa Cruz: 1Department of Biomolecular Engineering, 2Department of Molecular, Cell, and Developmental Biology, 3Department of Computer Science

April 2, 2020 Abstract

According to the United Nations, hunger affects approximately 10.6% of the world’s population (Food and Agriculture Organization of the United Nations, 2019). In an attempt to combat world hunger, both the Bill and Melinda Gates Foundation and Heifer International are integrating chicken farming into developing nations. These efforts are inhibited by the highly virulent Newcastle Disease Virus (NDV). Due to the lack of a cure or treatment, the only preventable measure against the disease is vaccination. The recommended vaccine, the LaSota type B1 lentogenic strain, must be transported and stored between 2-7 ◦C (Drugs.com, Updated: 2020-02-28). This strictly refrigerated distribution chain, termed the cold chain, poses an economic burden on developing nations. To ameliorate cold chain requirements we researched stabilizers produced by the world’s most resilient micro-animals, tardigrades. Tardigrade-speciﬁc intrinsically disordered proteins (TDPs) have the remarkable ability to protect, not only tardigrades, but yeast, bacteria, and enzymes from desiccation and temperature (Boothby et al. (2018), Piszkiewicz et al. (2019)). In this study, we characterized TDPs for use in a formulation to enhance the thermostability of the ND vaccine. We have begun evaluating a novel ND vaccine formulation and developed thermostability assays to compare our formulation against the commercially available LaSota type B1 NDV vaccine. We hypothesize our novel formulation can be applied to other vaccines to increase their accessibility and alleviate the cost of their cold chain requirements.

i Contents

Abstract ...... i List of Abbreviation ...... iv List of Figures ...... vi List of Tables ...... vii 1 Introduction ...... 1 1.1 Statement of Purpose ...... 1 1.2 History of iGEM at UCSC ...... 2 2 Global Impact ...... 2 2.1 The Cold Chain ...... 2 2.2 Current Newcastle Disease Vaccination Methods ...... 3 2.3 World Hunger ...... 4 2.4 Women Empowerment ...... 5 3 Outreach ...... 6 3.1 Fundraising ...... 6 3.2 Rotary Club ...... 7 3.3 Heifer International ...... 7 3.4 4-H Club ...... 8 3.5 Online Interactions ...... 8 3.6 iGEM Team Collaborations ...... 10 3.7 Girls in Engineering ...... 11 3.8 Santa Cruz County Schools ...... 12 3.9 Santa Cruz County Fair ...... 12 3.10 Bioethics ...... 12

ii 4 Presentations and Awards ...... 13 4.1 iGEM Giant Jamboree ...... 13 4.2 Cornucopia and Santa Cruz Tech Meetup ...... 14 4.3 Symposium For Undergraduate Research ...... 14 4.4 Annual Biomedical Research Conference for Minority Students (ABRCMS) 16 5 Scientiﬁc Background ...... 16 5.1 Tardigrades and Intrinsically Disordered Proteins ...... 16 5.2 Newcastle disease virus ...... 18 6 Methods ...... 18 6.1 Molecular Cloning ...... 19 6.2 Protein Production and Puriﬁcation ...... 20 6.3 Circular Dichroism ...... 21 6.4 Virus Propagation and Titration ...... 22 6.5 Novel ND Vaccine Formulation ...... 23 6.6 Hemagglutination Assay ...... 24 6.7 Plaque Assay ...... 25 6.8 Thermostability Testing ...... 25 7 Discussion and Results ...... 26 7.1 Plasmid Constructs ...... 26 7.2 Protein Production ...... 28 7.3 TDP Structural Dynamics ...... 35 7.4 Thermostability Assay Development ...... 42 7.5 Future Work ...... 46 8 Acknowledgments ...... 48

Bibliography 48

A Supplemental Information 56 1 Protocols ...... 56

iii List of Abbreviation

• ABRCMS - Annual Biomedical Research Conference for Minority Students

• APHIS - Animal and Plant Health Inspection Service

• CAHS - Cytosolic Abundant Heat Shock

• CD - Circular Dichroism

• CDC - Center for Disease Control

• CDFA - California Department of Food and Agriculture

• CSS - Cascading Style Sheets

• EH&S - Environmental Health and Safety

• EOP - Educational Opportunity Program

• FPLC - Fast Protein Liquid Chromatography

• GAVI - Global Alliance for Vaccines and Immunization

• GIE - Girls In Engineering

• HA - Hemagglutination Assay

• HIV - Human Immunodeﬁciency Virus

• HTML - Hypertext Markup Language

• IDP - Intrinsically Disordered Protein

• iGEM - International Genetically Engineered Machine

• JS - JavaScript

• KAN - Kanamycin

• KHD - Kinase, Ligase, DPN1

iv • LB - Luria Broth

• LEA - Late Embryogenesis Abundant

• ND - Newcastle Disease

• NDV - Newcastle Disease Virus

• PBSci - Physical and Biological Sciences

• PCR - Polymerase chain reaction

• PPE - Personal Protective Equipment

• qRT-PCR - Quantitative Reverse Transcription PCR

• SAHS - Secreted abundant heat shock

• SDS-PAGE - Sodium dodecyl sulfate Polyacrylamide gel electrophoresis

• SIV - Simian immunodeﬁciency virus

• STEM - Science, Technology, Engineering, and Mathematics

• SURU - Symposium for Undergraduate Research

• TDP - Tardigrade-speciﬁc IDP

• UCSC - University of California, Santa Cruz

• UNC - University of North Carolina

• UNICEF - United Nations Children’s Fund

• USDA - United States Department of Agriculture

• WHO - World Health Organization

v List of Figures

1 Violet the Vaccine Cover ...... 11 2 SURU Presentation Photo ...... 15 3 ABRCMS Photo ...... 16 4 pET28b Plasmid Map ...... 27 5 E. Coli Growth Curve ...... 29 6 SDS PAGE Results: CAHSD ...... 30 7 SDS PAGE Results: CAHSD Large Scale ...... 31 8 Large Scale Production of His-tagged Proteins ...... 32 9 His-tag Afﬁnity Chromatography ...... 33 10 CAHSD FPLC Puriﬁcation ...... 34 11 CAHS D FPLC Run Data ...... 35 12 CAHS D Melt CD Spectra ...... 36 13 CAHSD Melt and Post-melt Comparison ...... 38 14 CAHS 1 and CAHS D Tm Comparison ...... 39 15 SAHS10 Melt CD Spectra ...... 41 16 SAHS10 Tm Comparison ...... 42 17 HA Titer of our ND Vaccine Formulation ...... 44 18 Thermostability Testing ...... 45

A.1 Thermocycler Conditions ...... 63

vi List of Tables

1 Protein Name Translations ...... 19 2 SDM Primers ...... 20 3 qRT-PCR Reaction Parameters ...... 26 4 Protein Theoretical Molecular Weight and pI ...... 28

vii Introduction

1.1 Statement of Purpose

We intend to eradicate cold chain requirements for the Newcastle Disease (ND) vaccine and potentially all vaccines. ND virus (NDV) infects 80% of unprotected poultry and is considered to be the most devastating agricultural disease in rural areas (Morrison, 1998). It is an acute respiratory disease that causes lethargy, diarrhea, social isolation, and death (Miller, 2014). NDV is common across the globe with recurring outbreaks usually observed in Africa and Asia, sometimes two to three times a year(Bunn, 2020). The disease is spread from avian feces, respiratory discharge, and the reuse of soil in farms that have had previous outbreaks (Morrison, 1998). There has recently been an increase in the number of Newcastle disease cases in California. The United States Department of Agriculture (USDA) found 448 locations in California where chickens have been infected with NDV, primarily in San Bernardino and Riverside counties (USDA APHIS, 2019). The California Department of Food and Agriculture (CDFA) issued a statement about the epidemic which included maps of quarantined areas (CDFA, 2019). To prevent the spread of the virus, about 1.2 million birds in these quarantined areas have been killed, even if they have not exhibited symptoms of the disease (Cosgrove, 2019). ND can be devastating to those who rely on chickens for their food source. This can lead to the hunting and consumption of non-domesticated animals to acquire bushmeat as food for themselves and their families. Not only does consumption of bushmeat threaten the populations of exotic species, but it also leads to the spread of disease from animals such as bats and primates(Bunn, 2020). The consumption of bushmeat has led to zooanotic epidemics such as SARS-CoV of 2003 and possibly the current COVID-19 pandemic (World Health Organization (2020b) and World Health Organization (2020a)). Proper NDV vaccination is needed to combat against food insecurities and disease. As a team, we recognize these issues and are committed to address them through the development of our heat-stable vaccine formulation.

1 1.2 History of iGEM at UCSC

The International Genetically Engineered Machines (iGEM) competition is a nonprofit organization promoting problem solving and human practices through synthetic biology. Every year, more than 6,000 researchers from around the world head to Boston to participate in the yearly conference. At the conference, our project was judged on our human practices, scientific methods, website, and oral presentations. Bronze, Silver, or Gold medals were awarded to teams based on criteria from the iGEM website (iGEM, n.d.). Some criteria include: collaboration with other iGEM teams, adding BioBricks to the iGEM BioBrick registry, and working with communities that would be affected by the project’s results and the public to understand the overall issue. Since 2013, student-led teams at University of California, Santa Cruz (UCSC) have organized a project for the annual iGEM conference. At UCSC, iGEM grants a unique, hands-on educational experience to all members by fostering collaboration among various disciplines. Each iGEM project at UCSC has prepared students for graduate school, the scientific workforce, or starting their own companies. Vitrum, the UCSC 2019 team, is a collaboration of diverse undergraduate students who are applying their academic experiences to solve humanitarian issues through science and engineering.

Global Impact

2.1 The Cold Chain

The cold chain places requirements on every aspect of the supply chain and is difficult to maintain. Vaccines need to be stored at a certain temperature to maintain potency (World Health Organization, 2015). This requires specific shipping and handling parameters as vaccines are transported to different locations before administration. Each of these locations must have a constant source of power to keep a refrigeration system functioning 24 hours a day (World Health Organization, 2015). In addition, reliable transportation is required to ensure that vaccines arrive at their destination before they degrade. This strict chain is difficult for any country to maintain. It requires a comprehensive level of financing, infrastructure, and political will to distribute these

2 lifesaving vaccines. Not every country has the capabilities to guarantee this demanding cold chain. This leaves many resource constrained populations without vaccines and results in a reliance on poorly-funded nonproﬁt, NGO, or volunteer efforts. The World Health Organization (WHO) reported a 300% increase of measles cases in 2019, while in Africa the number of cases rose by 700% (World Health Organization, 2019b). Kagina and colleagues have speculated that this massive increase is due to the resources of nonproﬁts being directed toward Ebola prevention programs and away from measles vaccination initiatives (Kagina, 2019). This again highlights the fact that vaccine distribution has not been solved and requires further improvement. One such improvement could be the utilization of heat resistant vaccines allowing less resource constraints on storage and more focus on distribution.

2.2 Current Newcastle Disease Vaccination Methods

Chicken vaccines require very precise handling to prevent the decline of potency (Poultry Hub, 2020). When a farm receives a vaccine, it must immediately be placed in the specified storage conditions. According to the vaccination guidelines by the Poultry Hub organization, the recommended storage temperature for typical vaccines is below freezing while the dilutant must be kept at a temperature slightly above freezing (Poultry Hub, 2020). Our team is working with the LaSota type B1 lentogenic strain of the ND vaccine. This strain must be stored between 2 ◦C and 7 ◦C to maintain potency (Drugs.com, Updated: 2020-02-28). Chickens are administered vaccines through drinking water, feed, in-ovo, injection, and eye-drops (Poultry Hub, 2020). The ND vaccine we are working with can be administered in many ways, but the most common methods are through drinking water and aerosol spray (Drugs.com, Updated: 2020-02-28). Currently, chickens are vaccinated in-ovo for NDV to initiate flock immunity. However, immunity is not guaranteed throughout the chicken’s life, thus chickens need a ND vaccine booster every three months (Drugs.com, Updated: 2020-02-28). This illustrates the need for adequate storage of the vaccine for frequent use, but many countries don’t have access to high quality storage units. Removing or reducing the need for cold storage allows for farmers to focus on getting their chickens vaccinated rather than worrying about the efficacy

3 of the vaccine.

2.3 World Hunger

It is estimated that 852 million people globally are malnourished and have extreme vitadeficiencies (Muller¨ and Krawinkel, 2005). Malnourishment and hunger are the predominant factors contributing to the prevalence of disease and death in countries with low to middle income (Muller¨ and Krawinkel, 2005). Access to nutritional food should not be limited by geography, yet availability to these necessities is disrupted by politics, economics, and environmental stresses. Even with the progress made by humanitarian organizations, hunger remains a global issue. Many resource-constrained areas rely on international markets to supplement their inadequate food supplies, but international markets are limited by transport expenses and international trade rules (Elliott, 2015). In many parts of Africa and Asia, people are consuming bushmeat to alleviate hunger in their communities. Bushmeat is meat from non-domesticated animals such as local primates, bats, or snakes. Although bushmeat provides a food source and nutrients, there is a lack of education about the types of meat being ingested and the complications that go along with it. When humans consume primate species, there is a possible increase in the transmission of infectious diseases such as human immunodeficiency virus (HIV) (Asher, 2017). Ape species can carry simian immunodeficiency virus (SIV) which later is believed to have mutated in a human host to form HIV. The AIDS Institute believes SIV was most likely transmitted to humans through the consumption of bushmeat (The AIDS Institute, 2011). Other zooanotic viruses, such as the SARS-CoV epidemic of 2003, can arise from the consumption of animals which fall in the bushmeat category (World Health Organization, 2020b). An abundance of healthy chickens could decrease the spread of diseases from bushmeat to humans, reduce the threat to wildlife, and provide a reliable food source. The Gates Foundation vowed to donate 100,000 chickens to reduce poverty and hunger in resource-constrained areas (BBC News, 2016). Chicken meat and eggs provide a high quantity of protein, an adequate vitaB12 intake, and fatty acid content that is important for brain development (Pelleg, 2019). In addition to providing nutritional value, chicken-meat and eggs increase economic stability through trade (Lee,

4 2017). Vitrum is inspired to change the world through a heat-stable formulation of a Newcastle disease vaccine to protect chickens. By ensuring chicken ﬂocks are not infected with this disease, we can increase reliable food sources to people in rural areas.

2.4 Women Empowerment

Bailey et al. provided us with a candid depiction of Hoy Lin and her journey to becoming a successful business woman. Hoy Lin was only an 8th grader in Cambodia when she dropped out of school to support her family by working in the rice ﬁelds. Her job demanded squatting and lifting while walking barefoot through dirt and waste. After a long day on the farm and taking care of her family, she had little motivation to start something bigger for herself. Then, Hoy Lin joined a group of eager farmers who started a co-op supported by Heifer International. From there, she began a successful business for herself. Recently, Hoy Lin built a shop where fellow farmers can buy fertilizer, feed, and other farming supplies. Due to her accomplishments, Hoy Lin can now provide her family with a greater income, more nutrient-rich foods, a larger home, and better clothes (Bailey, 2019). When women, like Hoy Lin, are empowered in agriculture, there is an increase in food products and food security. According to Feed the Future, there is also an improvement in other domains of empowerment like decision making, income, and leadership (Feed the Future, 2012). A World Bank article reported that one struggle women face is a lack of resources such as land and livestock, which leads to a low production of food. With the proper resources, women can improve their situation and maximize their economic opportunities (The World Bank, 2017). If women in resource-constrained areas have access to our heat-stable vaccine, then they can increase the survival rate of their chickens. Saving their chickens will increase their proﬁt because they will generate more food. A heat-stable Newcastle disease vaccine can help women in agriculture create economic stability.

5 Outreach

3.1 Fundraising

The UCSC iGEM team independently fundraises to pay for team and individual registration, travel and accommodations, lab supplies, and equipment for our experiments. Fundraising was an opportunity to present ourselves and our project professionally to a wide variety of people and organizations including biotechnology companies, the Santa Cruz community, and nonproﬁt organizations. The team created a crowdfunding page through UCSC to accept individual donations from friends, family, and the UCSC community. We also organized meetings with multiple UCSC departments such as Engineering, Biochemistry, Educational Opportunity Program (EOP), Physical and Biological Sciences (PBSci), and the different residential colleges. Because we have members from various STEM majors, we made connections with Dean Wolf of Baskin Engineering and Dean Koch of Physical and Biological Sciences. In addition, we received a generous donation from the Hymowitz Family Foundation to help support our research. We also had a meeting with George Spix, who was a generous donor of the 2018 UCSC iGEM team. That support was essential for building the lab we now use and for covering a signiﬁcant portion of our expenses. This process has educated us about the business aspects of a research project as well as the challenges that many companies face when interacting with investors and spreading awareness. Through fundraising, we have improved our public speaking skills, interacted with professionals, and learned how to connect our project to those that express the same passion for philanthropy and science.

6 3.2 Rotary Club

Rotary International is an organization that unites people from all different backgrounds to increase welfare, compassion, and peace around the world through their local communities (Rotary International, n.d.). Currently, there are over 1.2 million Rotary members and over 35,000 Rotary clubs worldwide that support following the Rotary principles of service, global impact, and leadership (Rotary International, n.d.). Each Rotary Club has speciﬁc charities that they are involved with, in which they organize events to raise money for such charities through their community outreach and support. Similar to Rotary club International, our principles and values include using our service and passion to help make a global impact. We were able to present our work at different rotary chapters such as Santa Cruz-Sunrise, Santa Cruz, Scotts Valley, and Aptos Rotary Club. The presentations were organized by Richelle Noroyan, the Community Relations Representative of UCSC. After our presentation at the Sunrise Rotary, we made a personal connection with a representative of Rotary International. This led to Rotary International asking if our team would like to collaborate on their conservation services in Kasese, Uganda. The feedback we received at the Sunrise Rotary presentation reassured us that our project will have an impact on many different communities. Our connection with the local Rotary Clubs has created a foundation for future UCSC iGEM team collaborations.

3.3 Heifer International

Heifer International is a nonprofit organization dedicated to ending hunger and poverty by sending livestock to underrepresented communities (Heifer International, 2019). Heifer trains small scale farmers to use environmentally friendly farming methods and how to create and operate a business. With this training, the farmers can educate other members of their communities, creating a more solid economic foundation. Heifer believes that instilling these farming practices will lead to a more resilient community with a positive impact on gender equality and a substantial reduction in poverty and hunger (Heifer International, 2019). Fortunately, Worldbuilders, which is a nonprofit organization that hosts fundraisers to inspire public donations in support of other nonprofits (Worldbuilders, 2020), graciously provided us with

7 the contact information of Heifer’s Director of Philanthropy, Jackie Finch. We set up a meeting with Jackie Finch and Dilip Bhandari, the Director of Program and Livestock Technology. During the meeting, they provided us with specific locations where our project would be most beneficial. They informed us about the refrigeration process abroad and how the cold chain is affected by it. They explained that transportation of vaccines in these remote areas is entirely reliant on an ice box. This is extremely unreliable and the vaccines can lose efficacy but the farmers would never know. Breaking the cold chain can provide farmers a dependable vaccine that they can use without fearing that it became inactive at some point. In addition, they explained how Heifer educates affected communities on the importance of vaccination and how to properly vaccinate chickens. Through this collaboration, we gained a better understanding of the real-world applications of our project.

3.4 4-H Club

The 4-H Club is a national youth development organization focused on empowering children around the world (4-H, n.d.). Their mission is to help younger generations build life-long skills and become thoughtful and contributing members to society. Local programs use hands-on projects to allow their members to gain experience in the ﬁelds of health, science, agriculture, and civic engagement. We reached out to the 4-H clubs in Santa Cruz, Monterey, Aptos, and Watsonville with the goal of forming a partnership for summer 2020. We hope to talk to the children enrolled in the program about Vitrum and the importance of vaccinating animals. Explaining the importance of vaccination practices will help fulﬁll the goals of 4-H and spread awareness of Newcastle disease.

3.5 Online Interactions

We used multiple forms of social media to reach a larger audience and gain additional perspectives on our project. We used Instagram to share our project with other iGEM teams and our friends. Through Instagram, we advertised fundraising events around campus that provided us an opportunity to talk directly to students and faculty about our project. We discovered a Facebook page dedicated to ending virulent Newcastle disease in Southern

8 California. The purpose of this page is to educate its members about virulent Newcastle disease based on information from the CDFA. The members of this page update each other about Newcastle outbreaks and quarantines, kill zones, eradication goals, and more through information provided by the CDFA. By joining this page we were able to interact directly with farmers and everyday citizens who own chickens and face the challenges created by NDV. We wrote a post for this Facebook group describing the main goal of our project while focusing on the human practice aspect. We encouraged the members to tell us their experiences with NDV and why a heat-stable Newcastle disease vaccine would be beneficial to them. One of the members told us that the CDFA does not endorse vaccination, but veterinarians and biologists recommend it. Through personal communication with the community and government officials, we found that the USDA does not inform those with backyard flocks about vaccination because they use non-industrial flocks as an indicator for the spread of NDV. Therefore, commercial flocks vaccination is not required unless an outbreak of NDV occurs near their factory. From this outreach effort, we were able to connect to an online community that is affected by NDV who educated our team about the CDFA and USDA regulations for vaccinating chickens. Our website, or wiki page https://2019.igem.org/Team:UCSC, is an extension of our outreach campaign. It is personalized for the respective project of our team. The wiki page is hosted on iGEM servers, so future teams can be inspired by and continue to build off of past projects. Our website allows the public to learn about our project and our team. As we present our work to different audiences, they can use our website to stay updated with our progress. In addition, the website is used to educate viewers about the ethics of our project. Even if we cannot meet with people in person, we want them to feel connected to our purpose and values. For our human practices, we aim to find communities around the world that would be affected by our project. The website connects us to the people and organizations in those communities. It has also led to collaborations with other iGEM teams, allowing us to learn about their projects and we can work together to meet some common goal.

9 3.6 iGEM Team Collaborations

The iGEM program allows teams to connect with one another to share protocols and share information about similar projects to get a different perspective. We attempted to collaborate with as many 2019 iGEM teams as possible. We organized a meetup in Santa Cruz with the Brown-Stanford-Princeton 2019 iGEM team. We presented our projects to each other in preparation for the iGEM jamboree in Boston. The meetup was another opportunity to practice explaining our project. After each presentation, we provided feedback on the presentation, content of the project, and stage presence. We also had a Q&A session. We asked questions about the challenges each team faced and how they overcame those challenges. This furthered our discussions about human practices and the ethics of our projects. This collaboration provided meaningful insight about which aspects of our project can be improved and how we can implement those changes. The Vienna 2019 iGEM team challenged us to write a six-page story about our project that is geared toward all audiences. The purpose was to practice communicating our project to any audience, even children. We wanted to make the goal of our project easy to understand and appealing. Our main character was Violet the Vaccine; a superhero who has the power to turn into gelatin when exposed to extreme heat. In the book, she travels to areas that need her help and ends with her in a clinic, ready for administration. Through this collaboration, we hope the public will gain a better understanding of all aspects and applications of our project. We sent our book to the Society for the Prevention of Cruelty to Animals (SPCA) of Monterey County, a nonproﬁt shelter that tends to the needs of all animals and educates the public about humane practices (SPCA, n.d.). The SPCA’s youth camps used our book as an educational tool to teach children about the cold chain.

10 Figure 1: The cover page of our book that we sent to both the Vienna iGEM team and the SPCA. Please see our website for the full book https://2019.igem.org/Team:UCSC/ Collaborations.

3.7 Girls in Engineering

Girls In Engineering (GIE) is a five-day free program at UCSC for seventh and eighth-grade girls (GIE, n.d.). This program encourages interest in computer science and engineering through hands-on activities. The girls learn to program robots to pick up and carry objects, create 2D animations, build computer games, and tour labs at UCSC (GIE, n.d.). An important aspect of our human practices focuses on women empowerment. Our team contacted GIE with the hopes of empowering young girls to pursue a career in STEM. GIE toured our lab where they were able to see a research project in progress. The females of iGEM visited their classroom and discussed the benefits and challenges of being a female in the STEM field. We hoped to be an example that it is possible to be a female and be successful in STEM. Seeing these young girls already taking an interest in STEM inspired us to stay motivated about our own project.

11 3.8 Santa Cruz County Schools

We presented our iGEM project to various schools within the Santa Cruz County School District. The classrooms we visited ranged from elementary to high school. We designed a simplified outline of our project in the hopes that students in any grade would understand our research. The students were able to ask questions which we greatly encouraged. In addition to discussing our research, we emphasized the importance of representation in the STEM field using our diverse team as an example. We hoped to encourage students of any age, race, culture, etc to pursue a career within the STEM field. As a couple of team members grew up in Santa Cruz and attended some of the schools we visited, we were proud to show the students that their local university is a viable option for their academic future. Most importantly, our presentations allowed us to outreach to the younger generations and inspire them to find their passion(s).

3.9 Santa Cruz County Fair

Our team presented each day at the Poultry Show at the Santa Cruz County Fair. Through a local 4-H Club, we contacted Terry Reeder, who organizes the Poultry Show. At the show, we presented an informational booth about Newcastle disease and the project. We created flyers to educate the general public on the importance of recognizing symptoms of NDV in chickens. We emphasized how vaccinating their chickens against the virus can reduce the number of outbreaks in California. We paired these flyers with posters and the consistent presence of our team members. We answered questions and listened to stories of how Newcastle disease has affected the lives of many Californians. You can find our posters on our website https://2019.igem.org/ Team:UCSC/Public_Engagement

3.10 Bioethics

The 2019 iGEM team worked with the UCSC course BME 80G: Bioethics in the 21st Century: Science, Business, and Society which introduced us to some ethical questions regarding our research. After presenting our project to the class, the students wrote a short paper discussing the bioethics of our project and discussed their opinions and feedback on our work.

12 Through our discussions, it was brought to our attention that our method of increasing heat stability by shielding the virus could be used as a bioweapon. Viruses that are currently viable for two hours could potentially be protected for months with our heat stable method. It is important to understand both the positive and negative effects of one’s research, which includes being aware of the potential dangers it can bring forth. We asked questions addressing a multitude of consequences to build a stronger project. An important topic of our discussion was our use of embryonated chicken eggs. We wanted to make sure our work with these eggs was ethical and avoided animal testing. There are many regulations and policies in place to lessen the negative impact of our work. To understand the regulations of animal testing, we can speak with experts from the Center for Disease Control (CDC). The use of embryonated chicken eggs requires us to follow local, state, and federal guidelines on ethical testing. We were told that chickens are only considered animals when they hatch at day 21. With this information, we would have to ﬁnish working with the eggs before this day. This changed the course of our research to make sure our protocols follow these regulations, and we began developing in vitro assays to avoid the use of chicken embryos. As we continue to make progress on our project, we will remind ourselves of the ethics of each step we take. Regardless, our overall goal is to make a positive impact on farming communities in developing nations by practicing ethical procedures.

Presentations and Awards

4.1 iGEM Giant Jamboree

At the 2019 iGEM Giant Jamboree, we had the opportunity to present our work to protect the Newcastle disease virus with IDPs. Jonah Pierce, Liana Beld, Preet Kaur, Rita Ousterhout, and Shayan Vahdani gave a 20 podium presentation covering our human practices, outreach, scientiﬁc research, and BioBrick contributions to our judges and audience. In additon, our entire team gave poster presentations to our judges and teams interested in our work. By convincing the judges of our BioBrick, or IDP, characterization and validation, our collaborative approach, and our human

13 practices oriented problem solving accomplishments, we were awarded a silver medal. Our silver medal is tied with the highest honor in UCSC iGEM history!

4.2 Cornucopia and Santa Cruz Tech Meetup

To share our work with the UCSC community we presented at a variety of events both on and off campus. On campus, we set up a booth at the annual Cornucopia to get students interested in the current year’s iGEM and to potentially recruit for the next year’s team. We emphasized the hands-on research opportunities the iGEM program provides and the impacts previous years teams have had. The Santa Cruz New Tech Meetup is a technology-based event that connects companies with the community of Santa Cruz. The UCSC iGEM Team was fortunate enough to be a part of the 10 startups and companies that presented a 1-minute pitch of our purpose and project. As a team of young professionals in biotechnology, we found this opportunity beneﬁcial to our work because it gave Vitrum credibility and connected our project with people in our community. It taught us how to pitch an idea as a viable startup to other companies and learn about what other engineers are doing in the ﬁeld.

4.3 Symposium For Undergraduate Research

The Symposium For Undergraduate Research (SURU) is an annual event hosted by UCSC aimed at supporting and encouraging undergraduate involvement in research through both poster and oral presentations. Martina Pederson and James Hahn gave an oral presentation on our project at the 2019 SURU (Figure 2). They received a presentation award for the best oral presentation.

14 Figure 2: Team members James Hahn and Martina Pederson giving a podium presentation at the 2019 Symposium for Undergraduate Research at University of California, Santa Cruz. Their presentation resulted in the Best Oral Presentation Award.

15 4.4 Annual Biomedical Research Conference for Minority Students (ABRCMS)

The Annual Biomedical Research Conference for Minority Students is one of the largest scientiﬁc conferences tailored to underrepresented minorities in the science, technology, engineering, and math ﬁelds. One of our team members, Preet Kaur, was accepted to present at the 2019 ABRCMS conference in Anaheim, CA (Figure 3). Her participation in a poster presentation under the Engineering, Physics, and Mathematics discipline resulted in a Best Presentation award.

Figure 3: Our team member, Preet Kaur, was accepted to present at ABRCMS 2019. Her poster presentation to judges, as shown in the left picture, resulted in a Best Presentation Award in the Engineering, Physics, and Mathematics discipline.

Scientiﬁc Background

5.1 Tardigrades and Intrinsically Disordered Proteins

Tardigrades are a phylum of micro-animals, renowned for their ability to survive various extreme conditions such as desiccation, high pressure, extreme temperatures, and even the vacuum of space (Horikawa, 2012). Our research had originally focused around the tardigrade’s

16 ability to survive desiccation and its ability to function normally after re-hydration (Horikawa, 2012). We later began researching the tardigrade’s ability to survive temperatures of up to 151 ◦C (Piszkiewicz et al., 2019). One reason tardigrades can survive these extreme conditions is due to the various unique proteins they express called Tardigrade-specific intrinsically disordered proteins (TDPs) (Boothby et al., 2018). We are studying two types of TDPs: cytosolic abundant heat shock proteins (CAHS) and secreted abundant heat shock proteins (SAHS). As IDPs, these proteins do not have a regular three dimensional structure (Boothby et al., 2018). The proteins vary between fully structured and partially unstructured and are not able to be imaged by standard techniques such as X-ray crystallography. Their molecular mechanism is not completely understood, however Boothby et al. (2018) hypothesized that these proteins form a glass-like structural matrix around proteins and organelles to shield from protein-protein interactions and support structural integrity. We hypothesize the mechanism is electrostatic in nature, in which the IDPs bind to pockets of opposite charge on the protein capsids of virions and form a glass-like structural matrix during desiccation. This glass-like matrix structure may provide protection from desiccation and thermal degradation (Boothby et al. (2018) and Piszkiewicz et al. (2019)). Piszkiewicz et al. (2019) has shown successful protection of the enzyme lactate dehydrogenase with TDPs, and we hope to expand upon these results by protecting virions in a vaccine. Specifically, (Piszkiewicz et al., 2019) showed CAHS 94063 and CAHS 89226 maintained about 60 % of the activity of desiccated lactate dehydrogenase after exposure to 95 ◦C for 10 min (Piszkiewicz et al., 2019). Also, (Boothby et al., 2018) showed that after heating various desiccated CAHS recombinant yeast that there was no decline from initial survival percentages until after 60 ◦C was reached. (Boothby et al., 2018) hypothesized that the TDPs reach a glass transition phase after 60 ◦C, and that this transition decreased the survival percentage (Boothby et al., 2018). The specific molecular mechanism by which these proteins confer heat tolerance and desiccation tolerance remains to be elucidated. CAHS and SAHS are respectively localized in most intracellular compartments or extracellularly, however they are not present in the mitochondria (Yamaguchi et al., 2012). Mitochondrial integrity is necessary for cell survival, but the protective molecules of the mitochondria are still being discovered (Tanaka et al., 2015). RvLEAM, a Group 3 LEA protein,

17 was the ﬁrst conﬁrmed mitochondrial LEA proteins in tardigrades and IDPs improved the hyperosmotic tolerance of human HEp-2 cells (Tanaka et al., 2015). LEA proteins have been found in various plants and non-plant organisms which are tolerant to desiccation and abiotic stress, such as cold temperatures and salt. LEA proteins can bind directly to protein surfaces and replace coordinated water or order water molecules around the associated macromolecules as ”molecular shields” to reduce denaturation and inactivation of proteins (Mertens et al., 2018).

5.2 Newcastle disease virus

Newcastle disease virus, or Avian Avulavirus 1, is a negative sense, single-strand, RNA virus of the Paramyxoviridae family (Miller et al., 2013). Newcastle disease causes birds to exhibit respiratory failure, nervous system problems, diarrhea, lesions, and eventually, death (Miller et al., 2013). While Newcastle disease cannot be transferred to humans, studies have shown that it can cause conjunctivitis. It has been shown to occur speciﬁcally in poultry workers working with infected birds (Nelson et al., 1952). According to Merck and Co., a pharmaceutical company that produces vaccines for Newcastle disease, chickens are the most susceptible species to Newcastle disease while waterfowl are the least (Miller, 2014). Chickens shed the disease through their feces, exhaled air, and respiratory discharges (Miller, 2014). This shedding, combined with the fact that Newcastle disease can be highly virulent, can result in entire ﬂocks becoming infected in a matter of days. NDV is separated into three strains depending on how virulent the virus is. Velogenic is the most virulent, mesogenic is the second most, and lentogenic is the least (Miller, 2014). Due to the risks associated with Newcastle to fowl and humans alike, our team opted to work with the lentogenic strain of Newcastle disease. The lentogenic strain is used as a vaccine containing the live virus and is typically administered through feed, water spray, or eye drops (Miller, 2014). Administration of the vaccine causes chickens to be less susceptible to the disease, but the immunity decreases over time, resulting in a need for boosters (Miller, 2014).

Methods

Detailed protocols are included in Appendix A: Supplemental Information.

18 6.1 Molecular Cloning

Bacterial Transformations and Plasmid Library Preparation

We received 24 plasmids containing the genes of various IDPs from Dr. Roger Chang of the Silver Lab at Harvard University and one from Jonathan Eicher of the Pielak lab at University of North Carolina (UNC). This allowed us to forego designing our own plasmids and utilize cloning methods that recognized the promoters and terminators supplied to us. We worked with plasmids containing the DNA sequences for the following proteins: CAHS 107838, CAHS 94205, and SAHS 10, rvLEAM, Group 3 LEA 1, and CAHS 106094. The plasmids were transformed into DH5-α cells following manufacturer guidelines (New England Biolabs, n.d.a). We then prepared index plates, liquid cultures, and performed colony PCR on the transformed cells. We extracted the plasmids from the liquid cultures using the Zymo Zyppy Miniprep kit (Zymo Research, 2019).

UniProt Name Lab Name CAHS 107838 CAHS 1 CAHS 94205 CAHS D CAHS 106094 CAHS 2 rvLEAM rvLEAM Group 3 LEA 1 LEA 1 SAHS 10 SAHS 10

Table 1: This table denotes the names of our IDPs by UniProt and the naming nomenclature used in our lab.

Site-directed Mutagenesis

In order to substitute the Flag tag with a 6X His tag, we designed primers using the NEBaseChanger tool (New England Biolabs, n.d.b). After an initial failure due to an unintended hairpin sequence, primers were redesigned. Site-directed mutagenesis was performed with the Q5 polymerase system and touchdown PCR following manufacturer protocol (New England Biolabs, n.d.b). PCR was followed by ligation using NEB’s KLD reaction mix. Ligated mutant plasmids were transformed into DH5-α cells to create plasmid stocks. PCR products were sequenced to verify that the correct mutations had occurred. The successfully mutated plasmids were

19 transformed into the T7 polymerase expressing E. Coli cell line, BL21 XJb(DE3) Autolysis cells (Zymo Research, 2019), for protein production. Primer sequences for SDM are located in Tabel 2.

Primers for 6X His-tag Substitution Tm: 56 ◦C fwd-Primer: 5’-caccaccatGATGATGATGATAAGATGATG-3’ rev-Primer: 5’-atggtggtgCATGGTATATCTCCTTCTTAAAG-3’ Primers for 6X His-tag and Enterokinase cut-site Insertion Tm: 64 ◦C fwd-Primer: 5’-catgatgatgatgataagATGTCAGGGCGTAACGTG-3’ rev-Primer: 5’-gtggtgatggtggtgcatGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTC-3’

Table 2: This table contains the primer and sequences we used for 6X His-tag substitution or insertion on the 5’ end of the IDP insert sequence in our pET28b+ plasmids. Lower case regions indicate the new sequence being cloned. Upper case regions indicate primer binding region of the primer to the plasmid backbone. The lower set of primers was only used on CAHS D as it lacked both a His-tag and Enterokinase cut site.

6.2 Protein Production and Puriﬁcation

A growth curve was generated for our BL21 XJb(DE3) cells which indicated that mid-log phase occurred after roughly 3 hours of growth at 37 ◦C. From liquid cultures of transformed BL21 XJb(DE3) Autolysis cells, we inoculated 1L of LB-KAN and incubated until the mid-log growth phase at 37 ◦C. The inoculated cultures were induced with a final concentration of 1 mM IPTG. Protein expression was conducted at 18 ◦C and after eighteen hours, the culture was centrifuged at 4 ◦C and 2500 rpm for 30 minutes. The supernatant was removed and the cell pellets were stored at -20 ◦C until used for purification. The pellets were resuspended in 50 mM HEPES/NaCl buffer at pH 7.4. Both the pellet samples and the supernatant samples were placed in either a 95 ◦C water bath or later, an autoclave, on the dry cycle for 20 min. The samples were then incubated at room temperature for 30 min. Samples were centrifuged for 40 at 3000 rpm and the resulting supernatants were filtered using a 0.22 µm syringe filter. Immobilized metal affinity chromatography (IMAC) was first performed following manufacturer’s guidelines with a cobalt column using gravity flow (ThermoFisher Scientific, n.d), but was later performed using a fast protein liquid chromatography (FPLC) system. After the sample was allowed to flow through the column was washed with 10 column volumes of 10 mM

20 imidazole or until the absorbance at 280 nm declined close to zero. The protein was then eluted using increasing imdazole concentrations from 50 mM to 300 mM in steps of 50 mM. A gradient puriﬁcation protocol was used with FPLC, washing the crude extract for 30 with 5 mM imidazole mL at 1 min , followed by gradient elution where the concentration of imidazole was increased from mL 5 mM to 500 mM linearly for 30 at 1 min . Samples were collected into microcentrifuge tubes, and those that displayed elevated 280 nm readings during elution were run on SDS-PAGE gels according to Biorad’s guidelines (Laboratories, n.d.) and visualized using Coomassie Blue.

6.3 Circular Dichroism

Circular dichroism was used to study the effect of temperature on our TDPs’ secondary structure. The CD spectra of our TDPs were modeled to predict CAHS D’s, CAHS 1’s, and SAHS 10’s sensitivity to temperature and potential stabilizing characteristics. Our methods for CD spectra measurements and data analysis are describe below.

Measurements

Far-UV data for the thermal melts were measured on a J1500 Circular Dichroism Spectrophotometer (JASCO, Easton, MD) using a 0.05 cm path length quartz cuvette. The cuvettes were seated in a Peltier-controlled cell holder for thermal ramping experiments. Temperature ranges of 5–75 ◦C for CAHS 1, -10–80 ◦C for CAHS D, and 5–70 ◦C for SAHS 10 were used. Data (175-280 nm) was collected with a 0.1 nm step, 4 nm bandwidth, 4-s integration nm ◦C time, scan speed of 50 min , and a ramp rate of 0.6 min . Multi-wavelength spectra (2 scans/temperature) were accumulated at each temperature. After the CD spectra at the highest melt temperature was measured, the program was set to return to 65 ◦C where the reversibility of the protein was tested. Additional reversibility test points were chosen, such as -10, 5, or 20 ◦C depending on the protein. For the post-melt measurements, three CD spectra were collected at these temperatures and averaged.

21 Data analysis

Single-wavelength CD data was obtained by averaging 2 nm around the wavelength of interest. For example, the data at 222 nm were obtained from the multi-wavelength spectra by averaging from 221 to 223 nm at each temperature. The wavelengths chosen for analysis are hallmarks of the different secondary structures. Data collected as ellipticity (θ) in millidegrees (mdeg) was converted to molar ellipticity ([θ]) in deg ∗ cm2 ∗ dmol−1 by the following equation,

θ(MRW ) [θ] = l[P ]

MolecularW eight g where MRW is the mean residue weight, or #AminoAcids , in mol , l is the path length (cm), and mg [P ] is the concentration of protein ( mL ). To determine temperature midpoints of our TDPs at fixed wavelengths, we normalized [θ] to fraction folded (f) values between 0 and 1. Based on observed structural transitions with respect to temperature, the CAHS proteins’ folded state was assumed to occur at the lowest recorded temperature point, and the SAHS 10 protein’s folded state was assumed to be at the highest temperature point. CAHS and SAHS 10 proteins’ sigmoidal curves were fit using the Van Genuchten-Gupta model (Van Genuchten and Gupta (1993)) via the scipy.optimize.curve fit() package. Models are further described in Section 7.3.

6.4 Virus Propagation and Titration

In Vivo Propagation

We propagated the virus via embryonated chicken eggs using diluted commercial vaccine or our novel ND vaccine formulation. Embryonated chicken eggs were incubated at 37 ◦C and 40 % humidity and turned 3 times daily for 10 days. The eggs were intermittently candled to verify which eggs contained viable embryos. At day 10, we again candled the eggs to locate the embryos, membrane, and blood vessels. We sterilized the eggs with ethanol and injected diluted vaccine into the allantoic ﬂuid with 1-mm tuberculin syringes. The hole punctures were sealed, and the eggs were incubated for 65 to 70 hours. After incubation, the eggs were placed at 4 ◦C for 2 hours.

22 Allantoic ﬂuid was harvested and puriﬁed from the terminated embryos by following the protocol outlined in Current Protocols in Microbiology ((McGinnes et al., 2006)). The propagated virus samples were stored at -80 ◦C until required for viral RNA extraction.

In Vitro Propagation

We propagated the virus via chicken embryo ﬁbroblast cells (DF-1 cell line ATCC (2018)) using diluted commercial vaccine or our novel ND vaccine formulation. DF-1 cells were seeded at 250,000 cells per well on a six well plate using DMEM medium supplemented with 2 mM L- µg Glutamine, 50 mL Penicillin Streptomycin solution, and 10% Fetal Bovine Serum. Cultures were ◦ incubated at 37 C and 5 % CO2. On day 2 of culture, the monolayer of cells was replenished with µg propagation medium (DMEM + 2 mM L-Glutamine + 400 mL Trypsin) and infected with 0.01 multiplicity of infection (MOI). The MOI was estimated by assuming that the cells doubled every 24hr and that 1 HA unit (see Section 6.6) of our virus stock contained approximately 104 virus particles (W.-S. (2017)). Three days after infection, the supernatant was collected, and viral RNA was extracted. Our in vitro virus propagation protocol was developed from published methods by Jang (2011).

Viral RNA Extraction

We used the Qiagen RNeasy Mini Kit to extract RNA from the allantoic ﬂuid or supernatant samples (Qiagen (2019)). Extracted RNA was puriﬁed using the DpnI DNA digestion protocol found in Molecular Cloning: A Laboratory Manual (Green and Sambrook (2012)). Pure RNA was stored at -80 ◦C until use for qRT-PCR.

6.5 Novel ND Vaccine Formulation

Our purposed method for formulating a novel vaccine contains the LaSota type B1 lentogenic strain of NDV and Tardigrade-speciﬁc intrinsically disordered proteins. We used this formulation in thermostability testing to determine our TDPs ability to stabilize the ND vaccine. The formulation was created using ND virions from the commercial LaSota ND vaccine.

23 Lyophilized commercial LaSota type B1 lentogenic ND vaccine (Drugs.com (Updated: 2020- 02-28)) was resuspended in 10 mL of Ultrapure water, aliquoted, and stored at -80 ◦C for later use. For our novel formulation we would thaw a vial of ND vaccine stock on ice, add 1 HA unit of vaccine stock per test sample to a concentrated solution of a large scale produced TDP. The stock ND vaccine + TDP mixture was washed 3 times with cold PBS in a 10 kDa centrifugal concentration spin column at 3000 RCF for 30 min. The puriﬁed and concentrated ND virion + TDP mixture was split evenly for each test sample and dried via SpeedVac at medium heat for about 2 hr or until dry. The dried novel formulation was re-hydrated in cold water prior to its use in viral titer assays.

6.6 Hemagglutination Assay

We performed hemagglutination assays (HA) to determine the La Sota type B1 lentogenic Newcastle Disease virion hemagglutinin titer relative to virion stock positive controls. Our protocol was adapted from Protocols in Microbiology (McGinnes et al. (2006)) and “Diagnosis and Methods,” in Molecular Virology of Human Pathogenic Viruses (W.-S. (2017)).

Red Blood Cell (RBC) Preparation

We obtained whole chicken blood, added Alsever’s Solution in a 1:1 ratio for anti-coagulation, and centrifuged at 500 RCF for 10 minutes. Blood plasma, buffy layer, and overlaying erythrocytes were aspirated. We washed the solution 3 times by re-suspending in PBS with 2mM Penicillin/Streptomycin. Puriﬁed RBCs were centrifuged at 500 RCF, the pellet was resuspended in PBS with 2mM Penicillin/Streptomycin or AS-7 (Cancelas et al. (2015)) at ﬁnal concentration of 10 % pellet volume per total volume and store at 2-7 ◦C for 1 week or 42 days.

HA Titer

After mixed 2-fold serial dilution of samples in duplication and negative control with 1% RBC solution, a 96 round bottom well plate was covered in sterile wrap and incubated for 1hr at 2-7 ◦C. The HA titer of each sample was observed from the inverse of the last serial dilution that showed RBC agglutination.

24 6.7 Plaque Assay

We attempted to develop a plaque assay using DF-1 (chicken embryo ﬁbroblast) cells to determine the infectivity of our vaccine samples by following the protocol outlined in Current Protocols in Microbiology (McGinnes et al., 2006).

Overlay Medium

We ﬁrst mixed 38.5 mL of 1X Advanced DMEM (Gibco), 500 µL Pen/Strep 10X stock solution, 1 mL 200 mM L-glutamine, and 10mL stock tryptose phosphate broth and warm to 46 ◦C. Then mixed 1580 µL of the media we made with 20 µL of 0.05 % Trypsin (Gibco) and 400 µL of 1.5 % Nobel agar which was autoclaved and cooled to 46 ◦C. Trypsin is a necessary component of the overlay medium when working with lentogenic viruses. Trypsin cleaves the membrane fusion (F) protein for infection and propagation in the cell (Jang, 2011).

Cell Seeding

Chicken embryo fibroblast cells (DF-1 cell line ATCC (2018)) were seeded in 6-well tissue culture plates at 300,000 cells per well with 2-3 ml cell culture media. The plates were placed in a 5 % CO2 and 37.5 ◦C incubator until cells reached 80 % to 90 % confluence. Duplicate 10-fold serial dilutions of our vaccine samples, from 10−2 to 10−8, as well as negative (cell culture medium) and positive (stock ND vaccine) controls were added in the plates after we washed the cell monolayer with PBS. After incubation at 37.5 ◦C for 45 min, the virus containing dilutions were aspirated from the plate, and 2 ml of 46 ◦C overlay medium was added. The overlay medium set for 30at room temperature, then the 6-well plates were placed in a 5 % CO2 and 37.5 ◦C incubator. After 72 hours incubation, we removed the overlay medium and fixed the cells with 2 mL of methanol for 5 min. The cell were stained with 1:20 diluted stock Giemsa staining solution to help visualize the plaque forming units.

6.8 Thermostability Testing

Our novel ND vaccine formulation and virions extracted from our stock ND vaccine were compared for their thermostability. We used the stock vaccine as a positive control and PBS (In

25 vivo propagation and HA) or cell culture medium (Plaque assay and in vitro propagation) as negative controls. Our novel formulation was heat shocked at 56 ◦C for various time points in a water bath and immediately placed on ice and re-hydrated with cold Ultrapure water before virus quantiﬁcation and titration. Hemagglutinin activity and structural stability was investigated via HA titration. Infectivity and replication was investigated via plaque assay, in vivo virus propagation, and in vitro virus propagation. Plaque assay titration is reported as plaque forming units (PFU). The in vivo and in vitro propagation methods were followed by RNA extraction and quantiﬁcation of the NDV matrix protein RNA sequence using the Qiagen RNeasy Mini Kit and Quantinova qRT-PCR kit (Qiagen (2019) and Qiagen (2015)). Table 3 describes the primer and probes sequences, as well as the reaction set up we used in our qRT-PCR experiments. Primers and Probe for NDV M protein fwd-Primer 5’-AGTGATGTGCTCGGACCTTC-3’ rev-Primer 5’-CCTGAGGAGAGGCATTTGCTA-3’ Probe 6FAM-TTCTCTAGCAGTGGGACAGCCTGC-BHQ1 qRT-PCR Reaction Set-up 1X 45 ◦C 10 min 1X 95 ◦C 5 min 42X 95 ◦C 5 sec 60 ◦C 30 sec

Table 3: This table contains the primer and probe sequences we used for targeting the M protein RNA sequence of NDV, as well as the reaction set up for our qRT-PCR experiments.

Discussion and Results

7.1 Plasmid Constructs

All of our plasmids utilize the pET28b plasmid backbone (Figure 4). The pET28b plasmid contains a gene for kanamycin resistance and a T7 promoter and terminator. The sequences of the IDPs were E. coli codon optimized and the IDPs were cloned into the plasmid backbones using Gibson assembly. All of our plasmids were graciously provided by Dr. Chang and Dr. Piszkiewicz from the Silver Lab and the Pielak Lab. The plasmids we chose to work with were modiﬁed using site-directed mutagenesis. All of the plasmids except CAHS D originally contained an N-terminal FLAG-tag with an enterokinase

26 cut-site, but we substituted the FLAG-tag with a 6x His-tag. For CAHS D we used different primers to insert a 6x His-tag and enterokinase cut-site at the N-terminus (Figure 4). We used the NEBaseChanger tool (New England Biolabs, n.d.b) to design primers for site-directed mutagenesis. Our ﬁrst set of primers unfortunately contained a hair pin, leading to troubleshooting when lack of protein expression was observed. After isolating the issue a new set of primers was generated and SDM’s were redone successfully.

Figure 4: This ﬁgure shows the pET28b plasmid we used to express 6x His-tagged TDP, CAHS D, in E. coli cells. The pET28b plasmid backbone is the same for all of our plasmid constructs in this study. It contains a selection marker for Kanamycin resistance, and a T7 promoter and terminator. The insert containing the DNA sequence for our IDPs is located between the initiation site ”ATG” to the terminal stop codon. This ﬁgure was generated by Geneious Prime 2019.2.1. (https://www.geneious.com).

We initially transformed our plasmids into DH5-α for robust molecular cloning of our plasmids. After performing site-directed mutagenesis, we mini-prepped to isolate the plasmid for sequencing. We veriﬁed that the 6xHis-Tag was properly integrated by sequence alignment of our sequencing data with the expected mutant sequence. We transformed into BL21 Xjb (DE3) E.coli cells for production of proteins.

27 We calculated the molecular weights and theoretical isoelectric points of our IDPs using ProteinParam.py, a protein sequence analysis algorithm modiﬁed from Dr. David L. Bernick’s work (Table 4). The isoelectric points will help us determine whether the charge of the IDPs affects their ability to protect virus in future work.

His-Tagged IDP Molecular Weight (kDa) Theoretical pI CAHS 1 28.2 5.28 CAHS D 27.0 5.94 rvLEAM 32.2 9.48 LEA 1 40.8 5.04 CAHS 2 28.5 5.52 SAHS 10 15.6 8.94

Table 4: This table presents the theoretical molecular weight and pI of the TDPs focused on in this study.

7.2 Protein Production

Our pET28b plasmid backbone contains T7 promoter and terminator. Since T7 polymerase is not naturally occurring in E. coli, we used BL21 Xjb(DE3) Autolysis E. coli cells (Zymo Research, 2019). This is a strain of E. coli that produces the T7 RNA polymerase necessary for transcription of our genes of interest. We determined the optimal time for induction by creating a growth curve of BL21 XJb(DE3) cells containing the CAHS D plasmid (Figure 5). The curve depicts the lag-phase, log-phase, and

stationary phase of our cells. Figure 5 shows that the mid-log phase of our cells is at an OD600 of about 0.466 and occurs after about 3.75 hr of incubation at 37 ◦C with shaking at 250 RPM in µg liquid LB medium supplemented with 50 mL of kanamycin. We observed a dip in cell concentration at the 6hr time point, which may have been caused by insufﬁcient blanking or by cell death from a lack of oxygen. We noticed liquid cultures were capped too tightly when the 6hr samples were collected. The growth curve indicated that we should induce our liquid cultures with IPTG after

3.75 hr of incubation or at an OD600 of about 0.466 for optimal protein expression. To characterize the localization of our protein after expression and their heat solubility, we induced cells in 10 mL LB/KAN media with a ﬁnal concentration of 1mM IPTG. We collected samples hourly for six hours and analyzed the heat-soluble and insoluble fractions of the

28 BL21 xjb(DE3) Autolysis Cell Growth Curve 1.0

0.8

0.6 OD600

0.4

0.2

experimental data f(x) = a / (1 + b * exp(-cx))^(1/d) : a=0.931, b=-1.000, c=0.555, d=-0.190 mid-log phase (3.75hr, OD600 0.466) 0.0 0 5 10 15 20 Time (hr)

Figure 5: This figure illustrates the growth of liquid cultures containing our CAHS D transformed, BL21 derived E. Coli cells over 23hr of incubation. It appears that the lag-phase occurred from 0hr to 2hr, log-phase occurred from 2hr to 6hr, and stationary phase began at 6hr. The figure represents triplicate data (blue) fit to a Richards growth model (red). Black lines indicate the mid-log growth phase.

supernatant and the lysate (Figure 6). The results from SDS-PAGE indicated that CAHS D, SAHS 10, CAHS 1, and LEA 1 were heat-soluble (Only non-His-tagged CAHS D results are shown (about 26 kDa)). No bands were observed for rvLEAM and CAHS 2 in the soluble supernatant fractions so they were not used after this point. From this experiment we also determined that the 3 hr, 4hr, and 6hr samples had clear 26 kDa bands compared to the other samples. CAHS D’s band intensity at 6 hr relative to the 4 hr sample showed no apparent change, so we decided to end expression at 4hr for large scale production. There are proteins left in the insoluble fractions of lysate which could have resulted from insufficient cell lysis. It is also possible that the 95 ◦C purification step caused a portion of the produced IDPs to become insoluble. To see if we could push their heat solubility even further, we performed heat purification using the autoclave which reached a temperature of 121 ◦C and visualized the results using SDS-PAGE (Figure 8).

29 Figure 6: This ﬁgure shows SDS-PAGE results from the expression of CAHS D in BL21 derived cells. Non-induced negative controls show no 26 kDa bands as expected. Insoluble and soluble fractions of the supernatant(Green and Neon-Green) show no 26 kDa bands. Insoluble and soluble fractions(Red and Baby-Blue) of the lysate show over-expression of a 26 kDa bands. This indicates that CAHS D was not secreted to the media. The bands in the insoluble supernatant appear darker because the insoluble fraction was resuspended in the residual of the soluble fraction, which concentrated the samples. The soluble fractions of lysate appear to have the most pure and consistent results for the samples taken at 3hr, 4hr, and 6hr of induction. Expected CAHS D bands in red rectangles.

30 Since the characterization of CAHS D protein expression revealed consistent success of purification in the heat-soluble lysate after 4hr of IPTG induction, we proceeded to large scale production. For large scale production we induced 1L cultures of CAHS D transformed BL21 derived E. coli cells with a final concentration of 1mM IPTG for 4hr. The cell pellet was purified by heat solubility and a sample from the soluble fraction was tested by SDS-PAGE to verify the purity of CAHS D (Figure 7). The SDS-PAGE results showed a large 26 kDa band along with a less concentrated 15 kDa band. The 15kDa band may be small heat soluble proteins native to the E. coli cells or fragmented CAHS D proteins. Due to logistical difficulties with a 4hr induction we consulted Dr. Rebecca Dubois who suggested doing a longer induction and a lower temperature to slow down growth and possibly inhibit proteases. We attempted an 18hr induction at 18 ◦C and saw similar results to the 4 hour incubation and proceeded with that method going forward.

Figure 7: This ﬁgure shows SDS-PAGE of soluble lysate from large scale protein production of CAHS D at 4 hr. SDS-PAGE showed over-expression of a 26 kDa band along with a lighter 15 kDa band. Expected bands are in red rectangles.

To investigate the thermostable limits of CAHS D, CAHS 1, SAHS 10, and LEA 1 we heat shocked them in an autoclave. We produced these IDPs using our large scale protocol and puriﬁed each protein by heating at 120 ◦C in an autoclave for 20 min. Figure 8 shows the soluble fraction and pellet fraction of our autoclaved proteins. Each protein remained soluble after heat shock, suggesting an extremely high retention of thermostability. There was high recovery of CAHS D and CAHS 1, moderate recovery of SAHS 10, and low recovery of LEA1. Given these recoveries

31 we decided LEA1 was not expressed at a high enough level for future testing in our novel ND vaccine formulation. We also observed a peculiar range of bands appearing in different positions for each proteins soluble fraction. This may be caused by protein-protein interactions between our IDPs and E. coli proteins, which somehow maintains their presence in the soluble fraction. Or, perhaps the smaller bands are from premature termination of transcription or translation of our IDPs.

Figure 8: Large scale production and 121 ◦C heat solubility puriﬁcation of His-tagged IDPs. Expected bands in red rectangles.

To further purify our IDPs from any remaining contaminates we utilized an IMAC system with a cobalt column. This was necessary as extraneous proteins and a large amount of DNA contamination were observed after heat-purification. Samples were filtered using a 0.22 µm syringe to remove any left over cell debris. After running the sample on the column, the flow through was analyzed by SDS-PAGE to observe what fraction of our protein was bound (Figure 9). For CAHS 1 and CAHS D, a significant amount of our protein of interest did not bind the column. We hypothesized this may be due to column saturation, as our protein was highly expressed in E.coli. The sample was then washed with 10 mM imidazole to remove any potential contaminants. The concentration of imidazole in the wash buffer remained low as earlier SDS-PAGE gels showed excessive elution when washing with higher concentrations. Elution was

32 done with increasing concentrations of imidazole to maximize purity and concentration of the elution. The best elutions were used for circular dichroism and propagation experiments. Figure 9 shows the 100 mM elution samples containing the highest concentration of protein; however extraneous bands are still present showing that this process can be optimized further.

Figure 9: His Purification by Gravity Flow: CAHSD, SAHS10, CAHS1 Here we demonstrate the successful purification of the above proteins via affinity chromatography. Elutions were from 50 mM to 300 mM imidazole. 100 mM imidazole yielded the most protein so it was included. The additional bands may be proteins that were able to weakly bind the His column. Expected protein bands highlighted in red.

Later, an FPLC became available to the team thanks to the Akeson Lab. The FPLC allowed us to purify our proteins much faster and had the added beneﬁt of auto collecting our elutions. With the assistance of our TA Ryan we replaced the pumps, targeting chip, and learned how to use the necessary software. Multiple trial runs were attempted, conditions were optimized, and mL we managed to successfully elute our protein using a ﬂow rate of 1 min and an elution gradient from 5 mM to 500 mM imidazole. In Figure 10, selected wash and elution samples are analyzed using SDS-PAGE. The samples were selected using the change in UV absorbance dictated by the blue line in Figure 11. The UV absorbance is directly proportional to the amount of protein eluting

33 from the column. This allowed us to easily tell which samples had the highest concentration of CAHS D. In some samples we again saw a secondary band which could be a result of premature termination of translation or transcription of CAHS D or another protein.

Figure 10: CAHS D Purification using FPLC with His-tag Affinity Column: Figure shows the successful purification of CAHS D. Expected bands shown in red.

34 Figure 11: Puriﬁcation of CAHS D by FPLC: Run Data: Additional Data for the run of CAHS D on the FPLC. The black line is the ratio of our two buffers (A = 5 mM and B = 500 mM) and shows the increasing concentration of imidazole as the more of Buffer B is added. The blue line is the 280 nm UV absorbance, which shows when protein is being eluted and the red line is conductance. Sample number shown in upper horizontal axis. Elevated UV absorbance with samples 36-45 correspond to puriﬁed protein.

7.3 TDP Structural Dynamics

To further characterize our TDPs, we analyzed their structure and interpreted how their structure may inﬂuence their stabilizing properties. By understanding our TDPs’ structural stability at extreme temperatures, we can model the structural dynamics of our TDPs. We used this model for comparing each TDP and predicting their stabilizing potential of our ND vaccine formulation.

The structural dynamics of our aqueous TDPs, in 20 mM Na2HPO4 buffer (pH 7.4), were analyzed by circular dichroism (CD) in a range of temperature conditions. For both CAHS D and CAHS 1, as temperature increased, we observed a structural change from α-helix toward a disordered or open conformation (Figure 12). This structural change is indicated by the characteristic α-helix local minima of 208 nm and 222 nm transitioning to a single 200 nm local minimum, which is characteristic of disorder (Greenﬁeld (2006)). Interestingly, CAHS D’s α-helical spectra grew in magnitude as temperature dropped below 0 ◦C (Figure 12). The

35 formation of structure at freezing temperatures and the absence of ice formation suggest possible anti-freeze or amorphous glass-like properties of CAHS D. In the melt spectra of our CAHS proteins, we observed a conserved structural dynamic shift from α-helix to disorder in response to increased temperature.

CAHS D Melt CD Spectra

Temperature ( C) 2000 -10 -5 0 5 1500 10 15 20 25 30 1000 35 40 45 50 500 55 60 65 70

Molar Ellipticity 75 (deg*cm^2 / dmol) 0 80

500

1000

190 200 210 220 230 240 250 Wavelength (nm)

CAHS 1 Melt CD Spectra

3000 Temperature ( C) 5 10 15 20 25 30 2000 35 40 45 50 55 60 1000 65 70 75 Molar Ellipticity (deg*cm^2 / dmol)

1000

190 200 210 220 230 240 250 Wavelength (nm)

Figure 12: CAHS D Melt CD Spectra: CD spectra of CAHS D at temperatures ramping up from -10 ◦C to 80 ◦C suggest a temperature dependent shift from α-helical structure toward disordered structure. CAHS 1 Melt CD Spectra: CD spectra of CAHS 1 at temperatures ramping up from 5 ◦C to 75 ◦C also suggest a temperature dependent shift from α-helical structure toward disordered structure.

Next, we compared the CD spectra during the melt and 16hr after the melt to characterize the structural reversibly of our CAHS proteins. In Figure 13 we investigated the structural reversibility of CAHS D and CAHS 1. We observed that 16hr post-melt at 5 ◦C, CAHS D and CAHS 1

36 had partially reversed to their original α-helical structures. When the 16hr post-melt sample was re-heated to 20 ◦C and 60 ◦C, CAHS D and CAHS 1 shifted toward a disordered structure as previously observed. The 16hr post-melt CAHS D shifted further into a disordered conformation than originally observed. This supports that CAHS D never fully recovered from the previous melt. CAHS 1 showed similar disorder shifting 16hr post-melt as observed in the melt CD spectra. The melt and post-melt comparison of our CAHS proteins suggest that both proteins have reversible structure dynamics, however it appears that CAHS 1 has more robust structural recovery from denaturation. To compare the structural changes observed in our CAHS proteins, we overlaid their normalized CD spectra at ﬁxed wavelengths versus temperature (T ) (Figure 14). The CD spectra were normalized by converting the molar ellipticities ([θ]), at a ﬁxed wavelength and for each

temperature point recorded, to fraction folded (ff ) values using the following equation,

[θ]observedT − [θ]unfolded ffT = . [θ]folded − [θ]unfolded

The fully unfolded and fully folded molar ellipticities were assumed to equal the molar ellipticities observed at the lowest and highest temperature points recorded. This assumption was based on the CAHS proteins’ α-helix to disorder structural shift as temperature increased. We chose to compare fraction folded curves for the ﬁxed wavelengths at characteristic α-helix minima (222 nm and 208 nm) and 198 nm. Although the CD spectrum of an α-helix has a positive absorbance at 192 nm, we selected 198 nm data to avoid data compromised by high light absorption at 192 nm. The experimental data were ﬁt using the Van Genuchten-Gupta model (Van Genuchten and Gupta (1993)),

maxF olded f (T ) = , f T d 1 + ( T m )

where T m represents the temperature midpoint and d is an optimization for goodness of ﬁt. d was used to model sensitivity because it appears to correlate with the protein’s structural sensitivity to temperature, or steepness of the curve (Van Genuchten and Gupta (1993)). The Van Genuchten-Gupta model of CAHS D and CAHS 1 at 222 nm estimated temperature midpoints of 292.1 K (19.1 ◦C) and 301.2 K (28.2◦C), respectively (Figure 14). The d values

37 CAHS D Melt and Post-melt Comparison

Temperature ( C) 2000 5 20 60 16hr post-melt 5 C 1500 16hr post-melt 20 C 16hr post-melt 60 C

1000

500 Molar Ellipticity

(deg*cm^2 / dmol) 0

500

1000

190 200 210 220 230 240 250 Wavelength (nm) CAHS 1 Melt and Post-melt Comparison

3000 Temperature ( C) 5 20 60 16hr post-melt 5 C 16hr post-melt 20 C 2000 16hr post-melt 60 C

1000 Molar Ellipticity (deg*cm^2 / dmol)

1000

190 200 210 220 230 240 250 Wavelength (nm) Figure 13: CAHS D Melt and Post-melt Comparison: CD spectra comparison at 5 ◦C, 20 ◦C, and 60 ◦C during and post melt suggest partial reversibility of α-helical structure and increased sensitivity for the formation of disordered structure post-melt. CAHS 1 Melt and Post-melt Comparison: CD spectra comparison at 5 ◦C, 20 ◦C, and 60 ◦C during and post melt suggest partial reversibility of α-helical structure and consistent patterns of disorder structure formation as temperature increases. suggest CAHS D’s (d = 21.9) structure was less sensitive to temperature change than CAHS 1 (d = 51.6), however the T m indicates that CAHS D begins transitioning in structure at lower temperatures than CAHS 1. The trends observed at 222 nm are consistent with 208 nm and 198 nm, which further supports our observations of the loss of α-helical structure as temperature increases. This CD characterization of our CAHS proteins can be used to help us compare thermostability proﬁles of other TDPs and help us model the temperature limits these TDPs may

38 have for stabilizing our ND vaccine formulation.

CAHS Tm Comparison: 222nm 1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0 208nm Van Genuchten Gupta model: 1.4 f_f(T) = maxFolded / (1 + ((T / Tm) ** d)) CAHS 1 maxFolded=1.003, Tm=301.150, d=51.580 1.2 CAHS D maxFolded=1.329, Tm=292.126, d=21.923 1.0 fraction folded data fraction folded data 0.8 CAHS 1 maxFolded=1.027, Tm=302.603, d=44.773 CAHS D maxFolded=1.270, Tm=296.467, d=20.385 0.6 fraction folded data

Fraction Folded 0.4 fraction folded data CAHS 1 maxFold=1.018, Tm=302.116, d=85.488 [ø]obv - [ø]u / [ø]f 0.2 CAHS D maxFolded=1.289, Tm=293.991, d=22.059 0.0 fraction folded data fraction folded data 198nm 1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0 240 260 280 300 320 340 360 Temperature (K) Figure 14: Comparison of CAHS D’s and CAHS 1’s structural dynamics at wavelengths characteristic of α-helical minima (222 nm and 208 nm) and maxima (198 nm). The molar ellipticities were normalized to fraction folded values and plotted against temperature. The fraction folded versus temperature data was modelled as a function of temperature using the Van Genuchet- Gupta model. CAHS D and CAHS 1 had estimated temperature midpoints ranging from 292.1 K to 296.5 K (19.1 ◦C to 23.5 ◦C) and 301.2 K to 302.6 K (28.2 ◦C to 29.6 ◦C), respectively. d indicated temperature sensitivity of CAHS D’s and CAHS 1’s structure to range from 20.4 to 22.1 and 44.8 to 85.8, respectively.

Following our CAHS proteins’ characterization, we did a CD spectra analysis for SAHS 10 to characterize its structural dynamics. In Figure 15 we observed SAHS 10’s temperature dependent shift from disorder toward a type 1 β-turn structure. The shift toward type-1 β-turn structure can

39 be interpreted by the change from one local minimum close to 200 nm, indicative of disorder, to a new local minimum around 206 nm and a nπ∗ peptide transition around 225 nm (Perczel and Fasman (1992)). This suggest that SAHS 10 is gaining structure as temperature increases. SAHS 10’s formation of secondary structure at increased temperatures supports its thermophilic properties and its potential for stabilizing the ND vaccine at high temperatures. We investigated SAHS 10’s structural reversibility by comparing CD spectra from during the melt and 16hr post- melt at temperatures of 20 ◦C and 65 ◦C. We observed exaggerated reversibility of SAHS 10’s structure (Figure 15). SAHS 10 appeared more disordered at 20 ◦C post-melt and more type-1 β-turned at 65 ◦C post-melt, in comparison to the original melt data. To model SAHS 10’s structural sensitivity to temperature, we analyzed its fraction unfolded values versus temperature at ﬁxed wavelengths characteristic of type 1 β-turn CD spectra minima (225 nm) and nπ∗ peptide transition (200 nm) (Figure 16). The observed molar ellipticities, at a

ﬁxed wavelength and for each temperature point (T ), were normalized to fraction unfolded (fu) values by the following equation,

[θ]observedT − [θ]folded fuT = . [θ]unfolded − [θ]folded

We assumed the fully folded and fully unfolded molar ellipticities were equal to the molar ellipticities observed at the highest and lowest temperature points recorded. This assumption was based on SAHS 10’s disorder to type-1 β-turn structural shift as temperature increased. The fraction unfolded data was ﬁt using the Van Genuchten-Gupta model,

maxUnfolded f (T ) = . u T d 1 + ( T m )

SAHS 10’s structural dynamic model was used to extrapolate the maximum unfolded asymptote and estimate the temperature midpoint, where SAHS 10 has transitioned half way from a conformation of disorder to a conformation of type-1 β-turn (Figure 16). The estimated temperature midpoints were 271.9 K and 291.1 K (-1.1 ◦C and 18.1 ◦C) at wavelengths 225 nm and 200 nm respectively. Since the 200nm data is lacking information on the unfolded asymptote of the curve, the temperature midpoint at 225 nm is likely more accurate. The model determined

SAHS 10’s structural sensitivity to temperature (d225nm = 30.5) is between CAHS D’s (d222nm =

40 SAHS 10 Melt CD Spectra

Temperature ( C) 5 4000 10 15 20 25 2000 30 35 40 45 0 50 55 60 65 2000 70

Molar Ellipticity 4000 (deg*cm^2 / dmol)

6000

8000

10000

190 200 210 220 230 240 250 Wavelength (nm) SAHS 10 Melt and Post-melt Comparison

4000 Temperature ( C) 20 60 16hr post-melt 20 C 2000 16hr post-melt 60 C

2000

Molar Ellipticity 4000 (deg*cm^2 / dmol)

6000

8000

10000 190 200 210 220 230 240 250 Wavelength (nm) Figure 15: SAHS 10 Melt CD Spectra: CD spectra of SAHS 10 in temperatures ranging from 5 ◦C to 70 ◦C suggest a temperature dependent structural shift of disorder toward type 1 β-turn. SAHS 10 Melt and Post-melt Comparison: CD spectra comparison at 20 ◦C and 65 ◦C during and post melt suggest reversibility of SAHS 10’s structure and increased sensitivity, 16hr post- melt, for disordered and type-1 β-turn structure formation in response to decreased and increased temperature, respectively.

21.9) and CAHS 1’s (d222nm = 51.6). SAHS 10’s low sensitivity to temperature relative to CAHS 1 and SAHS 10’s thermophilic behavior further validates its potential as a thermostabilizer. CD spectra analysis of our TDPs is a promising method for modeling their thermostable properties at the structural level. The structural dynamic models of our TDPs suggest that SAHS 10, CAHS D, and CAHS 1 are promising candidates for stabilizing the ND vaccine for their respective thermophilic, anti-freeze, and high temperature midpoint characteristics. Repeated CD

41 SAHS 10 Tm Comparison: Van Genuchten-Gupta Model: 225nm f_u(T) = maxUnfolded / (1 + ((T / Tm) ** d)) maxUnfolded=1.223, Tm=291.927, d=30.512 1.2 fraction unfolded data 1.0 maxUnfolded=2.892, Tm=271.892, d=29.345 fraction unfolded data 0.8

0.6

0.4

0.2

0.0

200nm

Fraction Unfolded 2.5 [ø]obv - [ø]f / [ø]u 2.0

1.5

1.0

0.5

0.0 240 260 280 300 320 340 360 Temperature (K) Figure 16: Comparison of SAHS 10’s heat sensitivity shows protein structure transition from disorder structure toward β-turn structure as temperature increases. We analyzed the fraction unfolded at ﬁxed type-1 β-turn minima (225 nm) and nπ∗ peptide transition (200 nm) wavelengths versus temperature, and use the Van Genuchten-Gupta model to extrapolate the maximum unfolded asymptote, and estimate the temperature midpoint (T m) and sensitivity (b). spectra analysis are required to improve our models at lower temperature points, estimate the range of error for our CD spectra, and determine the reproducibility of our ﬁndings. In our future work, we plan to use these structural dynamic models to characterize and compare other potential stabilizers. Also, thermostability testing of our ND vaccine with the characterized TDPs, will provide essential information for validating and improving our predictions of a TDP’s stabilizing capacity.

7.4 Thermostability Assay Development

To assess our TDPs’ stabilizing potential for the LaSota type B1 strain of NDV we: designed, implemented, and optimized thermostability assays for virus quantiﬁcation. We used techniques to quantify hemagglutinin concentration, plaque forming units, and NDV genome replication. These techniques include: hemagglutination assay, plaque assay, and virus propagation followed by qRT-

42 PCR. Initial studies of our ND vaccine formulation were carried out with hemagglutination assays (HA). The HA assay quantifies concentration of agglutinating particles in a solution by observing the dilution at which agglutination of Sialic acid containing surface molecules, on red blood cells (RBCs), ceases to occur (McGinnes et al. (2006)). Agglutination is observed by the absence of pooled RBCs at the bottom of a round bottom or V bottom well. If RBCs are agglutinated, they form a lattice-like structure with the virus particles and spread apart from eachother. In the absence of viral particles, the RBCs pool together and form a red button shape at the bottom of the well (McGinnes et al. (2006)). The highest dilution with the observation of agglutination is the inverse of the HA titer. For example, if agglutination ceases to occur at a dilution of 1:125, then the HA titer is equal to 125. To identify the HA titer of our stock LaSota type B1 ND vaccine and our novel ND vaccine formulation we thawed stock vaccine, concentrated the vaccine with His-tagged CAHS D in a 10 kDa diafiltration column, dried the virion/CAHS D mixture at medium heat using a SpeedVac, and re-hydrated it to its original concentration for subsequent HA titration. The top two samples in Figure 17 show that our stock LaSota type B1 ND vaccine (”Stock NDV”) and our novel CAHS D + LaSota type B1 NDV virion formulation (”Stock NDV + TDP Dried”) have an HA titer of 125. This means our process of diafiltering and drying the ND virions in the presence of CAHS D has no effect on their agglutinating activity. The ”NDV + TDP Dried - TDP” sample label indicates our novel ND vaccine formulation with CAHS D which has been diafiltered in a 100 kDa column post re-hydration, to test the LaSota type B1 strain NDV hemagglutinin stability without the presence of CAHS D. ”NDV + TDP Dried - TDP” sample appears to have almost completely lost all of its agglutination activity. We observed ”NDV + TDP Dried - TDP” to partially agglutinate at the 1:25 dilution, for an HA titer of 25. This loss of agglutination supports the necessity of heat stabilizers in our formulation process. To compare the stabilizing capabilities of the stock LaSota type B1 NDV vaccine with our novel ND vaccine formulation, we dried the stock vaccine in our SpeedVac under the same conditions as our novel formulation. Figure 17 shows the re-hydrated stock vaccine (”Stock NDV Dried”) was almost able to maintain complete activity of agglutination through our drying process. We observed partial agglutination in the 1:125 dilution for ”Stock NDV Dried”, which is still used

43 to quantify it’s HA titer (McGinnes et al. (2006)); however, this may indicate that our CAHS D proteins have superior desiccation stabilizing characteristics compared to what is commercially used.

Figure 17: HA Titer of our ND Vaccine Formulation with CAHS D: Hemagglutination assay of our novel ND vaccine formulation (”Stock NDV + TDP Dried”) shows no loss in HA titer in comparison to fresh stock ND vaccine (”Stock NDV”). The dilution factor (DF) or HA titer is calculated by 25i, where i is indicated at the top of each column. PBS was used as a negative control for each sample replicate. Fresh stock vaccine (”Stock NDV”), our novel ND vaccine formulation post re-hydration (”Stock NDV + TDP Dried”, our novel ND vaccine formulation post re-hydration and virus purification with a 100 kDa column (NDV + TDP Dried - TDP), and stock vaccine post re-hydration (”Stock NDV Dried”) had HA titers of 125, 125, 25, and 125 respectively. These findings suggest that our diafiltration and drying process maintains stable hemagglutinin, and diafiltration without stabilizers diminishes HA titer.

Previous thermostability studies using the LaSota strain of NDV by Omony et al. (2016) showed HA titers of 0 after exposure to 56 ◦C for 15 min, but there was no investigation of the viruses ability to replicate after heat shock. We determined that the HA titer reaches 0 after only 1 min at 56 ◦C (Data not shown). Another in vitro method of vaccine titration we developed was a plaque assay. However, our result indicated that neither the diluted commercial vaccine (control) nor our novel ND vaccine formulation was able to form plaques on the plate of the chicken embryo ﬁbroblast cells (DF-1 cell line ATCC (2018)). After several failures, we gave up on this method. To investigate the effect of the 1 min heat shock at 56 ◦C on the viruses ability to

44 Thermostability Testing

NF41a NF41b NF43a NF43b NF44a 0.8 NF44b NDV8a NDV8b NDV9a NDV9b NDV50a 0.6 NDV50b Rn

0.4

0.2

0.0

20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 Cycle Figure 18: In vivo virus propagation comparison of our novel ND vaccine formulation and stock ND vaccine with prior 1 min heat shock at 56 ◦C. Results were inconclusive due to technical replicate errors and biological replicate inconsistencies. replicate, and to test whether our novel formulation with CAHS D could protect the viruses ability to propagate, we used in vivo virus propagation followed by qRT-PCR. Embryonated chicken eggs were inoculated with our novel formulation after re-hydration and our stock ND vaccine post heat shock. After two days of incubation the eggs were terminated and the allantoic fluid containing our propagated virus was collected. RNA was extracted from the allantoic fluid and the NDV matrix protein RNA sequence was targeted and quantified using qRT-PCR. Figure 18 shows that our qRT-PCR results were inconclusive. There were both technical replicate errors and biological replicate inconsistencies. The technical replicate errors may be caused by issues with the reaction chemistry, such as EDTA contamination from our DpnI digestion step in RNA extraction. An internal control will be necessary to include for future work with the assay to assure reaction chemistry is working properly. The biological replicate inconsistencies may be caused by our RNA extraction method of the variability between egg propagation efficiencies. A viral RNA specific extraction kit may improve RNA extraction methods and moving away from in vivo propagation toward in vitro propagation will likely improve the robustness of our virus propagation methods (Jang (2011)). In vitro propagation was tested to investigate its potential for reducing the variability of our biological replicates. Monolayers of DF-1 chicken cells were infected with an MOI of 0.01 by

45 our novel formulation and stock ND vaccine post 10 min at 56 ◦C heat shock. The results from this assay were also inconclusive due to a malfunction with the reaction set-up on our qRT-PCR machine. Although we did not conﬁrm the results of this assay, the in vitro propagation process proved itself to be much simpler and more efﬁcient for producing test samples. In vitro propagation is more cost effective for a large amount of samples. Also, in vitro propagation is a more ethical procedure for propagating virus since we will no longer have to work with embryonated chicken eggs, which have potential for life.

7.5 Future Work

Boothby et al. and Piszkiewicz et al. have supported their hypothesis that desiccated TDPs vitrify and act as protective shields against protein-protein interactions and structural degradation, which maintained percent survival or percent activity of tardigrades, S. Cerevisiae, E. coli, and lactate dehydrogenase (Boothby et al., 2018; Piszkiewicz et al., 2019). Therefore, we hypothesized our chosen TDPs would protect the activity of the Newcastle disease vaccine at higher temperatures than what is currently required for the LaSota type B1 NDV vaccine storage. We have supported our hypothesis of thermal resilience of TDPs using circular dichroism, which characterized distinct structural mechanisms for maintaining solubility at high temperatures and modelled the stabilizing potential of each TDP we successfully cloned. We will continue testing our hypothesis by continuing to optimize our thermostability assays. This is essential for comparing the thermostability of our novel vaccine formulation with the commercially available LaSota type B1 ND vaccine.

Continuation of Thermostability Testing and Assay Development

To improve our methods of in vitro NDV quantification we have decided to begin development of a Focus Forming Assay (FFA). FFAs are used when the virus has difficultly forming plaques due to slow replication or lack host lysis capabilities, as we observed in our plaque assays. FFAs works by staining the viral antigen with florescently labeled antibodies for quantification of infected cells (Wheelock and Tamm, 1961). We hope through FFAs, we can come up with a more reliable and viable assay.

46 In addition to testing formulations with CAHS D, SAHS 10, and CAHS 1, we plan to test to the protective properties of trehalose, ubiquitin, and Iron chloride precipitation. (Piszkiewicz et al., 2019) has shown promising results of ubiquitin stabilizing LDH at 95 ◦C. We will also test various combinations of IDPs to see if they have a synergistic or antagonistic effect. These comparisons will allow us to determine if IDPs or other proteins would be an effective thermostabilizer for vaccines.

Other Therapeutics

Every year, vaccines save millions of lives through the prevention of various diseases. Vaccines are still not accessible to everyone, but efforts from nonproﬁts such as Doctors Without Borders, Global Alliance for Vaccines and Immunization (GAVI), United Nations Children’s Fund (UNICEF), and the WHO are making universal access to vaccines a major part of their mission. According to the WHO, vaccines currently prevent approximately two to three million deaths each year (World Health Organization, 2019a). These organizations clearly believe that expanding immunization efforts will save more lives in the future. Although there have been vast improvements to the access of vaccines, the reliance on the cold chain is making global immunization an impossible goal. If successful, Vitrum will make the ND vaccine more accessible to people all over the world, thus allowing them to vaccinate their birds and feed their families. Our method of conferring thermostability for the ND vaccine may also be applied to protect other protein-based therapeutics. Access to vaccines will only be restricted by manufacturing and physical delivery as we do not expect our excipient to result in manufacturing delays or excessive cost increases. This application would effectively eliminate the requirement of the cold chain, allowing people to access vaccines and therapeutics that were previously limited to them. A major hurdle in regulation will be testing the toxicity and immunogenic response of our IDP’s in humans. This future testing seems hopeful, as a published abstract by the Pielak lab shows no changes in weight or behavior when CAHS D was injected into mice (Esterly et al., 2019). We look forward to the day when thermostable vaccines are the typical formulation used to provide protection against veterinary and human viral pathogens, including SARS-CoV-2.

47 Acknowledgments

First, we would like to thank our PI, Dr. David Bernick. He has consistently provided us with guidance and advice throughout our project. Thank you to our TA, Ryan Modlin, who provided us with advice on our project and trained us to use the lab equipment. Thank you to Eefei Chen, project scientist in the Department of Chemistry and Biochemistry at UC Santa Cruz, for collecting all of our circular dichroism (CD) data and for providing guidance with our CD analysis. We are thankful for the advice of our secondary PI, Dr. Rebecca Dubois. We would like to thank Dr. Pamela Silver, Dr. Roger Chang, and Priya Jani of the Silver Lab at Harvard as well as Dr. Thomas Boothby, Dr. Samantha Piszkiewicz, and Jonathan Eicher of the Pielak Lab at UNC for supplying us with their plasmids containing the IDPs. We would like to acknowledge Dr. Kelly Kemp and Priscilla Pingrey for their advice on cell culture protocols. Thank you to Bari Nazario of the UCSC Institute for the Biology of Stem Cells for providing us with cell culture equipment and guidance. We would also like to thank the UCSC Biosafety Committee who reviewed and approved our Biological Use Authorization and Zymo Research, New England Biolabs and Integrated DNA Technologies for supplying us with reagents for our experiments. Thank you to Dr. David Bunn and Dr. Rodrigo Gallardo of UC Davis who explained to us the impact our project would have and the logistical issues of the cold chain. We would like to thank Dean Alexander Wolfe, Dean Paul Koch, Provost Sean Keilen, Pablo Reguerin, Asia Valdivia, The Hymowitz Foundation and George Spix for their generous donations. Finally, we would like to thank those who funded us through our crowdfunding and other avenues. Without all of you, we would not be able to perform our research.

48 Bibliography

4-H (n.d.). About 4-H. Available at: https://4-h.org/about/what-is-4-h/. Accessed: August 26, 2019

Asher, C. (2017). Illegal bushmeat trade threatens human health and great apes. Available at: https://news.mongabay.com/2017/04/ illegal-bushmeat-trade-threatens-human-health-and-great-apes/. Accessed: July 1, 2019

ATCC (2018). Umnsah/df-1 atcc R crl-12203TM gallus gallus embryo. Available at: https: //www.atcc.org/Products/All/CRL-12203.aspx#documentation

Bailey, A. (2019). Toxic Masculinity Is No Match For Cambodia’s Female Farmers. Available at: https://www.heifer.org/join-the-conversation/blog/2019/May/ from-farm-to-market-in-cambodia.html. Accessed: August 27, 2019

BBC News (2016). Bill Gates launches chicken plan to help Africa poor. Available at: https: //www.bbc.com/news/world-africa-36487536. Accessed: July 30, 2019

Boothby, T. C., Piszkiewicz, S., Holehouse, A., Pappu, R. V., and Pielak, G. J. (2018). Tardigrades use intrinsically disordered proteins to survive desiccation. Cryobiology 85, 137–138. doi: 10.1016/j.cryobiol.2018.10.077

[Dataset] Bunn, D. (2020). Presentation on newcastle disease by dr. david bunn

Cancelas, J., Dumont, L., Maes, L., Rugg, N., Herschel, L., Whitley, P., et al. (2015). Additive solution-7 reduces the red blood cell cold storage lesion. Transfusion 55.3, 491–498. doi: 10.1111/trf.12867

49 CDFA (2019). Virulent newcastle disease. Available at: https://www.cdfa.ca.gov/ ahfss/animal_health/newcastle_disease_info.html. Accessed: July 26, 2019

Cosgrove, J. (2019). To stop a virus, California has euthanized more than 1.2 million birds. Is it reckless or necessary? Available at: https://www.latimes.com/local/lanow/ la-me-ln-virulent-newcastle-disease-outbreak-in-southern-california-20190607-story. html. Accessed: July 16, 2019

Drugs.com (Updated: 2020-02-28). Newcastle Disease Vaccine (B1 Type, LaSota Strain). Available at: https://www.drugs.com/vet/ newcastle-disease-vaccine-b1-type-lasota-strain.html. Accessed: 2020-03-31

Elliott, K. A. (2015). Food Security in Developing Countries: Is There a Role for the WTO? Available at: https://www.cgdev.org/publication/ food-security-developing-countries-there-role-wto. Accessed: June 24, 2019

Esterly, H. J., Crilly, C., Piszkiewicz, S., Pielak, G., and Christian, B. (2019). Toxicity and Immunogenicity of a Tardigrade Cytosolic Abundant Heat Soluble Protein. The FASEB Journal 33.1, lb341–lb341. doi:10.1096/fasebj.2019.33.1 supplement.lb341

Feed the Future (2012). Women’s Empowerment in Agriculture Index. Available at: http://ebrary.ifpri.org/utils/getfile/collection/p15738coll2/ id/126937/filename/127148.pdf. Accessed: August 18, 2019

Food and Agriculture Organization of the United Nations (2019). (in brief) the state of food security and nutrition in the world 2019: Safeguarding against economic slowdowns and downturns. Available at: http://www9.who.int/nutrition/publications/ foodsecurity/state-food-security-nutrition-2019-inbrief-en.pdf

GIE (n.d.). UCSC Girls In Engineering. Available at: https://gie.soe.ucsc.edu/ girls-engineering. Accessed: August 26,2019

50 Green, M. R. and Sambrook, J. (2012). Molecular Cloning: A Laboratory Manual (New, York: Cold Spring Harbor Laboratory Press), 4th edn.

Greenﬁeld, N. J. (2006). Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1.6, 2876–2890. doi:10.1038/nprot.2006.202

Heifer International (2019). Heifer International. Available at: https://www.heifer.org. Accessed: July 25, 2019

Horikawa, D. D. (2012). Survival of Tardigrades in Extreme Environments: A model for Astrobiology. Cellular Origin, Life in Extreme Habitats and Astrobiology 21, 205–217. doi: 10.1007/978-94-007-1896-8 12

iGEM (n.d.). iGEM Medals. Available at: https://2019.igem.org/Judging/Medals. Accessed: July 30, 2019

Jang, J. (2011). Validation of a real-time rt-pcr method to quantify newcastle disease virus (ndv) titer and comparison with other quantiﬁable methods. J. Microbiol. Biotechnol. 21.1, 100–108. doi:10.4014/jmb.1006.06006

Kagina, B. (2019). DR Congo’s Ebola crisis has led to children dying from measles. Available at: https://qz.com/africa/1665055/ dr-congos-ebola-crisis-has-led-to-children-dying-from-measles/. Accessed: July 23, 2019

Laboratories, B.-R. (n.d.). A guide to polyacrylamide gel electrophoresis and detection. Available at: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/ Bulletin_6040.pdf. Accessed: August 18, 2019

Lee, S. (2017). Chickens: A solution to global poverty? Available at: https://www.usglc. org/blog/chickens-a-solution-to-global-poverty/. Accessed: July 26, 2019

McGinnes, L. W., Pantua, H., Reitter, J., and Morrison, T. G. (2006). Newcastle Disease Virus: Propagation, Quantiﬁcation, and Storage. Current Protocols in Microbiology 1.1, 15F.2.1– 15F.2.18. doi:10.1002/9780471729259.mc15f02s01

51 Mertens, J., Aliyu, H., and Cowan, D. A. (2018). LEA Proteins and the Evolution of the WHy Domain. Applied and Environmental Microbiology 84.15. doi:10.1128/AEM.00539-18

Miller, P., Afonso, C., El Attrache, J., Dorsey, K., Courtney, S., Guo, Z., et al. (2013). Effects of newcastle disease virus vaccine antibodies on the shedding and transmission of challenge viruses. Developmental Comparative Immunology 41.4, 505–513. doi:10.1016/j.dci.2013.06. 007

Miller, P. J. (2014). Newcastle Disease in Poultry (Avian pneumoencephalitis, Exotic or velogenic Newcastle disease). Available at: https://www.merckvetmanual.com/ poultry/newcastle-disease-and-other-paramyxovirus-infections/ newcastle-disease-in-poultry. Accessed: August 26, 2019

Morrison, T. G. (1998). Paramyxoviruses, Infection, and Immunity. Encyclopedia of immunology 3, 1909–1916

Muller,¨ O. and Krawinkel, M. (2005). Malnutrition and health in developing countries. CMAJ : Canadian Medical Association Journal 173.3, 279–286. doi:10.1503/cmaj.050342

Nelson, C., Pomeroy, B., Schrall, K., Park, W., and Lindeman, R. (1952). An outbreak of conjunctivitis due to newcastle disease virus (ndv) occurring in poultry workers. Am J Public Health Nations Health 42.6, 672–678

New England Biolabs (n.d.a). New England Biolabs. Protocol guidelines from NEB 5-alpha competent E. coli. Available at: https://www.neb.com/products/ c2987-neb-5-alpha-competent-e-coli-high-efficiency. Accessed: July 26, 2019

New England Biolabs (n.d.b). Q5 Site-Directed Mutagenesis Kit Protocol (E0554). Available at: https://www.neb.com/protocols/2013/01/26/ q5-site-directed-mutagenesis-kit-protocol-e0554. Accessed: July 26, 2019

Omony, J., Wanyana, A., Mugimba, K., Kirunda, H., Nakavuma, J., Otim-Onapa, M., et al. (2016). Disparate thermostability proﬁles and hn gene domains of ﬁeld isolates of newcastle

52 disease virus from live bird markets and waterfowl in uganda. Virol J 13.1, 103. doi: 10.1186/s12985-016-0560-0

Pelleg, G. (2019). Eggs, chickens and the end of malnutrition. Available at: https://thepoultrysite.com/articles/ eggs-chickens-and-the-end-of-malnutrition. Accessed: July 14, 2019

Perczel, A. and Fasman, G. D. (1992). Quantitative analysis of cyclic -turn models. Protein Science 1.3, 378–395. doi:10.1002/pro.5560010310

Piszkiewicz, S., Gunn, K. H., Warmuth, O., Propst, A., Mehta, A., Nguyen, K. H., et al. (2019). Protecting activity of desiccated enzymes. Protein Science 28.5, 941–951. doi:10.1002/pro. 3604

Poultry Hub (2020). Vaccination. Available at: http://www.poultryhub.org/health/ health-management/vaccination/. Accessed: August 14, 2019

Qiagen (2015). Quantinova probe rt-pcr kit - qiagen. Available at: https://www.qiagen.com/us/resources/resourcedetail?id= b3a9e014-1f06-437e-aa57-27f1da361cd7&lang=en

Qiagen (2019). Rneasy mini handbook - (en) - qiagen. Available at: https://www.qiagen.com/us/resources/resourcedetail?id= 14e7cf6e-521a-4cf7-8cbc-bf9f6fa33e24&lang=en

Rotary International (n.d.). About Rotary International. Available at: https://www.rotary. org/en/about-rotary. Accessed: August 26, 2019

SPCA (n.d.). SPCA. Available at: https://www.spcamc.org. Accessed: July 30, 2019

Tanaka, S., Tanaka, J., Miwa, Y., Horikawa, D. D., Katayama, T., Arakawa, K., et al. (2015). Novel mitochondria-targeted heat-soluble proteins identiﬁed in the anhydrobiotic tardigrade improve osmotic tolerance of human cells. PloS One 10.2, e0118272. doi:10.1371/journal.pone.0118272

53 The AIDS Institute (2011). Where did HIV come from? Available at: https://www. theaidsinstitute.org/education/aids-101/where-did-hiv-come-0. Accessed: August 26, 2019

The World Bank (2017). Help Women Farmers ’Get to Equal’. Available at: https://www.worldbank.org/en/topic/agriculture/brief/ women-farmers-getting-to-equal. Accessed: June 24, 2019

ThermoFisher Scientiﬁc (n.d). Instructions: HispurTM cobalt resin. Available at: https://www.thermofisher.com/document-connect/document-connect. html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets% 2FLSG%2Fmanuals%2FMAN0011581_HisPur_Cobalt_Resin_UG.pdf&title= UHJvZHVjdCBTaGVldDogIEhpc1B1ciBDb2JhbHQgUmVzaW4=

USDA APHIS (2019). Virulent Newcastle Disease (vND). Available at: https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/ animal-disease-information/avian/virulent-newcastle/vnd. Accessed: July 1,2019

Van Genuchten, M. T. and Gupta, S. (1993). A reassessment of the crop tolerance response function. Journal of the Indian Society of Soil Science 41.4, 730–737

W.-S., R. (2017). “diagnosis and methods,” in molecular virology of human pathogenic viruses. Elsevier , 47–62doi:DOI:10.1016/B978-0-12-800838-6.00004-7

Wheelock, E. and Tamm, I. (1961). Enumeration of cell-infecting particles of newcastle disease virus by the ﬂuorescent antibody technique. The Journal of experimental medicine 113(2), 301– 316. doi:10.1084/jem.113.2.301

World Health Organization (2015). Module 2: The vaccine cold chain. Available at: https: //www.who.int/immunization/documents/IIP2015_Module2.pdf. Accessed: July 20, 2019

World Health Organization (2019a). Immunization coverage. Available at: https://

54 www.who.int/news-room/fact-sheets/detail/immunization-coverage. Accessed: July 23, 2019

World Health Organization (2019b). New measles surveillance data for 2019. Available at: https://www.who.int/immunization/newsroom/measles-data-2019/ en/. Accessed: July 23, 2019

World Health Organization (2020a). Qa on coronaviruses (covid-19). Available at: https: //www.who.int/news-room/q-a-detail/q-a-coronaviruses

World Health Organization (2020b). Who — sars (severe acute respiratory syndrome). Available at: https://www.who.int/ith/diseases/sars/en/

Worldbuilders (2020). Worldbuilders. Available at: https://worldbuilders.org/ our-story/history/. Accessed: July 4, 2019

Yamaguchi, A., Tanaka, S., Yamaguchi, S., Kuwahara, H., Takamura, C., Imajoh-Ohmi, S., et al. (2012). Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade. PLOS ONE 7.8, e44209. doi:10.1371/journal.pone.0044209

Zymo Research (2019). Zyppy Plasmid Miniprep Kit. Available at: https: //files.zymoresearch.com/protocols/_d4019_d4020_d4036_d4037_ zyppy_plasmid_miniprep_kit.pdf. Accessed: July 26, 2019

55 Appendix A

Supplemental Information

Protocols

Additional Protocols may be found through citations or on our website https://2019. igem.org/Team:UCSC/Experiments

Protocol 1: Production of Luria Broth

Purpose: Production of bacterial growth media for agar plates and liquid cultures

Materials

• Tryptone

• Yeast Extract

• Sodium Chloride

• 70% Ethanol Solution

• Kanamycin

• Deionized Water (DI Water)

• Precision Balance

56 • Autoclave and Bin

• Thermometer

• 2L beaker

• Hot gloves

• Tinfoil

Conditional:

• Sodium hydroxide

• Agar

• 100mm x 15mm Petri Dishes

Methods: Amount Made = 1L Adapted from Molecular Cloning: A Lab Manual by Green and Sambrook (Green and Sambrook, 2012).

1. Weigh out, then add 10g of tryptone, 5g of yeast extract, and 10g of sodium chloride to a 2L beaker.

2. Add 1000 mL of DI water.

3. Optional: Add 950 mL of DI water then test the pH with a pH meter before adjusting with sodium hydroxide until the pH is 7.0. Then add DI water until the total volume is 1000 mL. This will tell you the exact pH of the solution, but we have found that the pH is normally very close to 7.0 anyway and small variations in pH have not affected bacterial growth on our plates.

4. Cover the top of the beaker with tin foil to prevent boiling over in the autoclave.

5. Put the 2L beaker in an autoclave bin and and autoclave on the liquid cycle for 20 minutes.

57 6. After removing from the autoclave with hot gloves, add a thermometer to the solution and allow to cool to 55◦C (If you do not have a thermometer, a good indicator the solution is cool enough is if you can touch and hold it for 10 seconds)

7. Add 50 mg of kanamycin (KAN) to the solution and mix

Conditional Methods for Agar Plates:

1. Before autoclaving add 15 g of agar

2. After adding kanamycin pour approximately 15 mL of media into a 100 mm by 15 mm petri dish.

3. When pouring multiple plates, stack subsequent plates on top of each other to prevent condensation.

4. Let set approximately 30 minutes at room temperature before storing in a refrigerator (4◦C) for up to one month.

5. Test the antibiotic in the plates by plating an antibiotic resistant strain on one plate and a non-antibiotic resistance strain on another and incubate overnight at 37◦C before checking for growth.

Analysis If your agar plates set then you added agar properly to it, if not you can use the media for liquid cultures. Note that you cannot autoclave it again as that will destroy the kanamycin. For your control test, if you see growth on the antibiotic resistant plate and no growth on the normal strain plate your antibiotic is functioning correctly.

Protocol 2: High Efﬁciency Transformation of NEB 5-alpha Competent E. coli Cells

Purpose: The goal of this protocol is to insert a plasmid into an E. coli cell.

Materials:

• PPE

58 • Sterilizing solution: ethanol

• Sterilized petri dish

• 20 µL, 200 µL, and 1000 µL pipettes and pipette tips

• 0.2 mL of NEB 5-alpha competent E. coli cells

• 25 mL of room temperature (25◦C) SOC outgrowth medium

• Plasmid to transform from blotting paper

• Sterilized exact-o-knife

• 1.5 mL Eppendorf tube

• Ice

• Ice bucket

• Heat Block (42◦C)

• Shaking Incubator (37◦C, 250 rpm)

• 2 LB/KAN plates

Methods: Adapted from New England Biolabs. Protocol guidelines from NEB 5-alpha competent E. coli (New England Biolabs, n.d.a).

1. Thaw a tube of 0.2 NEB 5-alpha competent E. coli cells on ice until ice crystals disappear. Carefully pipette 50 µL of cells into a transformation tube, then gently mix by ﬂicking. Put on ice.

2. On a sterilized petri dish, use a sterilized exact-o-knife to cut a 1 cm x 1 cm square of the plasmid blotting paper. Place square in transformation tube, then ﬂick tube 4-5 times to mix cells and DNA. Do not vortex.

3. Place transformation tube on ice for 30 minutes. Do not mix.

59 4. Heat shock at 42◦using a Heat Block for exactly 30 seconds. Do not mix.

5. Immediately place on ice for 5 minutes. Do not mix.

6. Use a 1000 µL pipette to add 950 µL of room temperature SOC into the transformation tube.

7. Place the transformation tube in an incubator at 37◦C and 250 rpm for 60 minutes.

8. Before plating, label the two LB/KAN plates with initials, date, LB, and KAN. Label one 20 µl and the other with 150 µL. Pre-warm plates in incubator at 37◦C.

9. Remove transformation tube from incubator and mix cells by ﬂicking or inverting the tube.

10. Pipette 20 µL from the transformation tube onto the 20 µL plate and 150 µL onto the 150 µL plate.

11. Store media plates in incubator overnight at 37◦C. Alternatively, incubate at 30◦C for 24-36 hours or at 25◦for 48 hours.

Analysis: Check media plates the next day for results. If successful, the media plates will have white colonies throughout. The plate with 150 µL should have more colonies then the 20 µl plate. If unsuccessful, the plates will not have any growth and the protocol must be repeated.

Protocol 3: Index Plating of Transformed E. coli Cells and Liquid Cultures

Purpose: To take individual E. coli cell colonies from the original transformation plate to another plate that is marked with a grid. This makes sure you are drawing from a monoclonal colony and helps with performing colony PCR. Liquid cultures will be used to grow E. coli Materials:

• LB/KAN plates with transformed E. coli colonies

• PPE

• Unused LB/KAN Plate

• Inoculation loops

60 • 1.5 mL Eppendorf tubes

• Sterile Water

• Permanent Marker

Methods: Adapted From Molecular Cloning: A Lab Manual by Green and Sambrook (Green and Sambrook, 2012).

1. Label each Eppendorf tube (1-9), and draw a 3x3 grid on the LB/KAN plate’s lid. Number each box (1-9).

2. Pipette 10 µL of sterile water into 9 labeled Eppendorf tubes, be sure the water is located at the bottom of each tube. If the water is on the side of the tube, brieﬂy vortex the tube.

3. Take an inoculation loop and pick up an isolated E. coli colony from your LB/KAN plate. The colony should not be touching any other colony on the plate. Do not reuse the same inoculation loop.

4. Place the E. coli colony in the sterile water.

5. Pipette 1 µL of the mixture onto a fresh LB/KAN plate in the numbered box that correctly corresponds to the label on the Eppendorf tube.

6. Spread the cells diagonally across the box. Make sure they stay in their own box.

7. Incubate the agar grid plate at 37◦C for 12 hours at 250 rpm.

8. the remainder of the E. coli solution can be used to start a 10 mL liquid culture by pipetting into 10 mL of liquid LB/KAN media in a 50 mL centrifuge tube and incubating overnight at 37◦C.

Analysis: Check media plates the next day for results. If successful, the media plates will have white colonies in each box. If unsuccessful, the media plates will not have any growth, or will be growing into each others boxes. If this is the case, the protocol must be repeated. For liquid cultures, if the media has become cloudy you were successful.

61 Protocol 4: Colony PCR

Purpose: To quickly and efﬁciently screen for plasmid inserts in transformed E. coli cell.

Materials:

• PCR tube

• DI H2O

• Transformed E. coli colonies

• PCR Mastermix (Contains One Taq)

• 10 µM Forward primer solution

• 10 µM Reverse primer solution

Methods: Adapted From Molecular Cloning: A Lab Manual by Green and Sambrook (Green and Sambrook, 2012). For a 50 µL reaction:

1. Pipette 23 µL of sterile water into a PCR tube.

2. Touch the tip of a pipette to an isolated colony of transformed E. coli cells and add to the PCR tube.

3. Pipette 25 µL of PCR Mastermix to the PCR tube.

4. Pipette 1 µL of Forward primer and 1µL of Reverse primer into the PCR tube. Primers degrade quickly and must be kept at a low temperature, so add primers last and put them back in a -20◦C freezer after use.

5. Run PCR using the thermocycler with the conditions in A.1.

Analysis: Remove sample from the thermocycler. Refrigerate for future analysis by gel electrophoresis

62 Figure A.1: X refers to the annealing temperatures of your primers which vary based on primer length and GC content. The elongation time also varies on what polymerase you are using and the length of DNA you are replicating, so make adjustments as necessary.

Protocol 5: Production of TBE and Gel Electrophoresis

Purpose: Making TBE for use in gel electrophoresis to determine the length of a DNA fragment.

Methods:

• 500 mL Erlenmeyer Flask

• ELGA H2O

• Tris Base

• Boric Acid

• EDTA

• Agarose

• Gel casting tray

63 • Electrophoresis Box

• Gel Imager

• 1 L Erlenmeyer Flask

• 125 mL Erlenmeyer Flask

• Hot gloves

• SYBR Safe DNA Gel Stain

• NEB Ladder

• Paraﬁlm

• Gel Loading Dye

• DNA sample

• Power supply

Methods: Adapted From Molecular Cloning: A Lab Manual by Green and Sambrook (Green and Sambrook, 2012). Making 10x TBE (500 mL):

1. Obtain a clean 500 mL Erlenmeyer Flask

2. Add 400 mL of ELGA H2O to the Flask

3. Dissolve 54 g of Tris Base [tris(hydroxymethyl)aminomethane], 27.5 g of Boric Acid, and 3.75 g of EDTA into the ﬂask.

4. Swirl the solution (5min) until no more clumps can be visible.

5. Fill the ﬂask up to 500 mL with ELGA H2O. Swirl again to dissolve all the clumps.

6. Store at room temperature. Do not place in refrigerator.

64 Making a 1% Agarose Gel for Electrophoresis of Plasmids:

1. Pre-cool the gel casting tray by placing at 4◦C.

2. Obtain a clean 1 L Erlenmeyer Flask

3. Mix 100 mL of 10X TBE with 900 mL of ELGA H2O in the 1 L ﬂask. (Only do this if there is no other 1X TBE available. The same TBE can be reused for many gels if it is saved.)

4. Make sure there is enough 1X TBE to be used for both the gel and the gel box. You do not want to mix different batches of buffer when pouring the gel and covering it.

5. Obtain a clean 125 mL Erlenmeyer Flask

6. Mix 0.75 g of Agarose with 75 mL of TBE in the ﬂask.

7. Microwave for 45 seconds. Use hot gloves to swirl the ﬂask.

8. Put back into the microwave for another 45 seconds. Use hot gloves to swirl the ﬂask. When microwaving, watch the ﬂask very closely. Once bubbles start forming, stop the microwave immediately. Let cool in microwave (5 min).

9. When temperature has reached about 55◦C, add 7.5 µL of SYBR Safe DNA Gel Stain and swirl to mix. (1µL to 10 mL of gel mixture).

10. Pour all 75 mL of agarose gel into the pre-cooled gel tray. Make sure the well comb is already in place. Pour slowly to prevent bubbles. Use a pipette tip to move bubbles to the side of the gel. Do this right after pouring, before the gel has a chance to set.

11. Let sit at room temperature for 20 minutes.

12. Fill gel box with 1X TBE until gel is covered.

Loading Samples and Running Agarose Gel:

1. Fill up an ice box and place 1 kb NEB ladder and Purple gel loading dye (6X) inside.

65 2. Cut a strip of parafilm. Separate the parifilm into different sections, depending on how many samples will be loaded into the gel. Use a sharpie to create the separations and label each section (a 3cm x 3cm square for each sample will suffice).

3. Add 1 µL of purple loading dye onto a square. Add 5 µL of the NEB ladder on top of the dot of purple loading dye and use the pipette to mix the two (5-10 up/down). Load the ladder into the ﬁrst well.

4. Add 1 µL of purple loading dye to 4 more sections of paraﬁlm. (Do not wait long to add the DNA sample as the dye will eventually adhere to the paraﬁlm).

5. Add the corresponding 5 µL of the DNA on top of the purple loading dye and use the pipette to mix the two (5-10 up/down). Load the 6 µL sample onto gel immediately. Repeat for rest of samples. (Remember to change pipette tips to avoid contamination. Or cleanse the same tip in TBE buffer before use.) Mark down which sample is going into each well.

6. Run the gel at 120 V until the dye line is approximately 75-80 % of the way down the gel. A typical run time is about 1-1.5 hours. You should see bubbles immediately, and the dyes moving across the gel after 10 minutes if everything is working properly.

7. Turn off the electrodes and carefully remove gel box. Place in tray and transport to Gel Digital Imaging System.

8. Use the imager to photograph the gel under the white light setting and use the software to annotate the gel bands/wells.

9. Save the image onto the lab computer and backup onto Google drive. After this is done, the gel can be thrown away in the Biohazard waste bin.

Analysis: To determine the length of the DNA compare the band to the ladder to get an estimate of its size. If you have no bands at all it is possible you didn’t add either the loading dye or the SYBR safe gel stain. If you have a smudge your DNA may be contaminated.

66 Zymo Miniprep for Puriﬁcation of plasmid DNA

Purpose: Puriﬁcation of Plasmid DNA from E.coli for Storage and Future Use.

Materials:

• 10 mL E.coli liquid culture

• PPE in accordance with BSL1 standards

• Ethanol

• Wash Buffer

• Zyppy Wash Buffer

• Lysis Buffer

• Neutralization Buffer

• Centrifuge

• Zymo-Spin IIN column

• Collection Tube

• 1.5 mL microcentrifuge tube

• Endo-Wash Buffer

• NanoDrop

Methods: The following methods were adapted from the Zymo Research Zyppy Plasmid Mini Prep Kit Protocol (Zymo Research, 2019). The following methods should be performed at room temperature (≈ 25◦C.)

1. Centrifuge the 10 mL liquid culture at 1000 rpm for 20 minutes before discarding the supernatant. Re-suspend the pellet in 600 µL of water and pipette up and down to mix.

67 2. Add 100 µL of 7X Lysis Buffer (Blue) and mix by inverting the tube 4-6 times, then incubate for 1-2 minutes. Do not vortex. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue. This indicates complete lysis.

3. Add 350 µL of cold Neutralization Buffer (Yellow) and mix thoroughly. Do not vortex. Neutralization is complete when the sample is entirely yellow and a precipitate has formed.

4. Centrifuge for 2-4 minutes at 11,000 x g.

5. Place a Zymo-Spin IIN column in a Collection Tube and transfer the supernatant from step 4 into the Zymo-Spin IIN column. Avoid disturbing the pellet containing cell debris as this will contaminate your sample with foreign DNA.

6. Centrifuge the Zymo-Spin IIN for 15 seconds at 11,000 x g.

7. Discard the ﬂow-through and return the Zymo-Spin IIN column to the same Collection Tube. Ensure the ﬂow-through does not touch the bottom of the column.

8. Add 200 µL of Endo-Wash Buffer to the column and centrifuge for 30 seconds at 11,000 x g

9. Add 400 µL of Zyppy Wash Buffer to the column. Centrifuge for 1 minute 11,000 x g.

10. Transfer the column into a clean 1.5 mL microcentrifuge tube then add 10 µL of Zyppy Elution Buffer 2 directly to the column matrix and let stand for one minute at room temperature.

11. Centrifuge for 30 seconds to elute the plasmid DNA.

Analysis: Analysis should be performed using a NanoDrop which will tell you the concentration of DNA in the solution as well as the amount of protein and salt in the solution. This will show you if the DNA is still contaminated or if it is pure. If the DNA is contaminated with protein you could attempt to run this protocol again to remove more of it or you could throw away the DNA and start a new culture. If there is a high salt content then you should not perform electroporation on these plasmids as the high salt content will cause the electricity to arc. Finally, if the DNA concentration

68 is above 200 ng/mL it could be that the chromosomal DNA from the bacteria contaminated the DNA and then it should be thrown out.

Site-directed Mutagenesis

Purpose: To remove or insert pieces of DNA into a plasmid via ﬂagged primers. Materials:

• Q5 Hot Start Master Mix

• 10 µM Forward Primer

• 10 µM Reverse Primer

• Template DNA (1–25 ng/µL)

• Nuclease-free water

Step 2:

• PCR Product

• 2X KLD Reaction Buffer

• 10X KLD Enzyme Mix

• Nuclease-free Water

Methods: This protocol was adapted from the New England Bio Labs Site-Directed Mutagenesis Protocol (New England Biolabs, n.d.b).

1. Obtain all materials and place on ice.

2. Add 12.5 µL of Q5 Hot Start High-Fidelity 2X Master Mix to each PCR reaction tube (Pre- label all PCR reaction tubes). Then, add 9 µL of Nuclease-free water to each tube as well. Keep on ice when not adding reagents.

69 3. Preheat the thermocycler by starting the saved thermocycler conditions and pausing the method immediately after starting. (This allows the thermocycler to preheat to the initial denaturation temperature)

4. Add 1 µL of template DNA (1-25 ng/µL) to the correct PCR tube. Make sure to not cross- contaminate.

5. Add 1.25 µL of 10 mM forward primer and 1.25 µL of 10 mM reverse primer to each tube. Flick tubes to mix and do a quick spin to get all the liquid to the bottom.

6. Run Touchdown PCR using the thermocycler with the conditions listed in A.1.

7. After PCR has completed, place the PCR product on ice.

8. Obtain more PCR tubes to conduct Kinase, Ligase, and DpnI (KLD) Treatment. Add 5 µL of 2X KLD Reaction Buffer, 3 µL of nuclease-free water, and 1 µL of 10X KLD enzyme mix to each KLD reaction tube.

9. Add 1 µL of each PCR product to their respective KLD reaction tube. Mix by ﬂicking and inverting. Incubate at room temperature for 5 minutes.

10. Use KLD treated PCR product and transform into 5-alpha E. coli. Refer to 10.1.2 Protocol 2: High Efﬁciency Transformation of NEB 5-alpha Competent E.coli Cells

11. Thermocycler conditions are shown in Figure A.1.

Analysis: Analyze products using gel electrophoresis on the PCR products or send the PCR products out for sequencing.

70 Protein Production: Induction of Protein Expression via IPTG (Day 1) Adapted in part from (Piszkiewicz et al., 2019) Purpose: Use IPTG to induce protein expression through E.coli BL21. Materials:

• PPE in accordance with BSL1 standards

• 20 ml LB-KAN medium

• 1000 µL overnight E.coli culture

• Incubator

• Spectrophotometer

• Isopropyl Beta-D-1-thiogalactopyranoside (IPTG)

• Centrifuge

• Freezer

Creating Liquid Cultures:

1. Follow Transformation protocol to transform desired protein plasmid into E. coli BL21 cells.

2. Take 3 colonies from the transformation plate and continue to make 3 liquid cultures and an index plate for those 3 colonies.

3. Incubate the liquid cultures at 37 C at 250 rpm overnight

Inoculation of Liquid Culture:

1. Obtain 3 sterilized 50mL Falcon Tubes. Fill each with 20mL of LB-KAN, warm liquid in incubator at 37 C.

2. Take out overnight cultures and ﬂick / invert to mix the tubes as the cells tend to settle at the bottom after storage.

71 3. Inoculate 1mL of each of the 3 overnight cultures into the 20mL LB-KAN tubes. Make sure the Falcon Tubes are properly labeled.

4. Place in Incubator at 37 C at 250 rpm. Shake in incubator for 3 hours. (The E. coli should reach mid-log phase)

5. Test OD600 at 3 hours. Record in notebook. (OD should be between 0.4 - 0.6).

Induction with IPTG:

1. Split each 20mL culture into two. You should end up with 2 x 10mL cultures (one is a control without IPTG induction and one with IPTG induction) for each of the 3 separate colony cultures, for a total of 6 tubes.

2. For each of the three IPTG induced tubes (NOT the control tubes) add 1.1mL of 10mM IPTG to reach a ﬁnal concentration of 1mM IPTG in the culture.

(a) Remove a 500 µl aliquot from each culture and place in 1.5mL reaction tubes. Centrifuge them at 1,000 rcf for 20 minutes. Remove the supernatant and save the supernatant separately in another tube.

(b) Freeze the cell pellets at -20◦C. These are zero time point samples.

3. Incubate all six tubes at 37◦C at 250 rpm for an hour.

4. Record the OD600 for each of the 6 tubes.

5. Take a 500µL aliquot from each culture (This is the 2hr time point). Put these aliquots in labeled 1.5mL reaction tubes. Put the cultures back into the incubator

(a) Centrifuge the aliquots at 1000 vcf for 20 min.

(b) Remove the supernatant from the pelleted tubes and transfer to new 1.5mL reaction tubes.

(c) End up with 6 pelleted (3 red [induced] & 3 blue [noninduced]) tubes and 6 Supernatant (3 green [induced] & 3 purple [noninduced]) tubes.

72 6. Incubate all six tubes at 37C at 250 rpm for another 2 hours.

7. Record the OD600 for each of the 6 tubes.

8. Take a 500µL aliquot from each culture (This is the 4hr time point). Put these aliquots in labeled 1.5mL reaction tubes. Put the cultures back into the incubator

(a) Centrifuge the aliquots at 1000 vcf for 20 min.

(b) Remove the supernatant from the pelleted tubes and transfer to new 1.5mL reaction tubes.

(c) End up with 6 pelleted (3 red [induced] & 3 blue [noninduced]) tubes and 6 SN (3 green [induced] & 3 purple [noninduced]) tubes.

9. Put the six culture tubes back into the incubator at 37C (250rpm) for 3 hours.

10. After 3 more hours (for a total of 6 hours) take a 500µL aliquot from each culture (This is the 6hr time point). Put these aliquots in labeled 1.5mL reaction tubes. Store the cultures at 4 C.

(a) Centrifuge the aliquots at 1000 vcf for 20 min.

(b) Remove the supernatant from the pelleted tubes and transfer to new 1.5mL reaction tubes.

11. End up with 6 pelleted (3 red [induced] & 3 blue [noninduced]) tubes and 6 SN (3 green [induced] & 3 purple [noninduced]) tubes.

12. You should end up with a total of 36 1.5mL reaction tubes. (12 from each 1hr, 3hrs, and 6 hrs) (3 red [induced], 3 blue [noninduced], 3 green [induced, & 3 purple [noninduced] for a total of 12 for each of the three time points)

13. Store the tubes at -20C.

73 Protein Production: Protein Purification and SDS Page (Day 2) Purpose: To get purified IDPs from E.coli cell lysis from induction of protein expression via IPTG by heat shocking and verified the plasmid through SDS-PAGE Material:

• Cell pellets and supernatants from induction of protein expression

• 25 mM Tris/HCl

• Water bath

• Centrifuge

• 2X SDS-PAGE sample buffer

• 1X SDS running buffer (see recipe)

• SDS stain solution (see recipe)

• SDS destain solution (see recipe)

• Mini protean TGX gels

• Power supply

• Electrophoresis Chambers

Procedure:

1. Turn on water bath and set to 95 C. (You can speed up this process by microwaving water and adding it to the water bath).

2. Resuspend the pelleted cells in 500µL of 25mM Tris/HCL. (Vortex to resuspend).

3. Put the pelleted samples AND supernatant samples (36 tubes total) in the water bath (95C) for 15 minutes. Use autoclave tape to secure the lid of the 1.5mL reaction tubes, you don’t want them to accidentally open.

4. Incubate at room temperature for 30 minutes.

74 5. Centrifuge at 16,100 rcf for 40 minutes.

(a) Remove supernatant from each tube and place into separate tubes.

(b) There should be a total of 36+36 = 72 tubes now.

i. 9 Red to 9 Baby Blue

ii. 9 Green to 9 Neon Green

iii. 9 Purple to 9 Black

iv. 9 Blue to 9 Orange

6. Mix all tubes via vortexing until the cells are fully suspended.

7. Take 6µL of each sample and put into new tubes

(a) Add 6µL of 2x sample buffer to each tube

(b) Vortex to mix, and tape caps closed, spin down to bottom

8. Load onto an SDS page cell (need gel, gel box, and running buffer)

(a) 2.5µL of Ladder

(b) 10µL of each sample

9. Run gel at 200V for 30 -45min (80% down)

10. Break the gels out of plastic using spatula. (pry open each side, run buffer over it, and peel off of plastic). Transfer into gel tray.

11. Wash gel with running buffer in the gel tray. (pour out buffer after the wash).

12. Add 25mL of coomassie blue stain to tray

(a) Cover tray with saran wrap and let sit at room temp while shaking for 30 - 40 min

75 (b) Can reuse stain. Pour back into stain storage container

13. Add 25mL of destain solution to tray, cover with plastic and shake at room temp overnight.

14. Next Day: Pour old destain out and add 35mL of new destain solution. Add rolled up paper towel to stain box. Shake at room temperature for 1 hour. Repeat another destaining after 1 hr. (For a total of 2 destainings)

15. Visualize gels in the white light box in the lab next door. (Akeson Lab)

(a) Can use clear gel tray, or cover light with saran wrap before transferring over gel on top.