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Maldi-Tof Mass Spectrometry and 16S Rna Approach for The MALDI-TOF MASS SPECTROMETRY AND 16S RNA APPROACH FOR THE IDENTIFICATION OF BACTERIA COLLECTED DURING THE PHOENIX MARS LANDER A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Biological Sciences By Devin L. Lachner 2020 SIGNATURE PAGE THESIS: MALDI-TOF MASS SPECTROMETRY AND 16S RNA APPROACH FOR THE IDENTIFICATION OF BACTERIA COLLECTED DURING THE PHOENIX MARS LANDER AUTHOR: Devin L. Lachner DATE SUBMITTED: Spring 2020 Department of Biological Sciences Dr. Wei-Jen Lin Thesis Committee Chair Biological Sciences Dr. Parag Vaishampayan Jet Propulsion Laboratory Dr. Wendy Dixon Biological Sciences ii ACKNOWLEDGEMENTS I want to thank Dr. Lin who took me into her lab when I was an undergraduate and gave me my first opportunity to perform research. This experience gave me direction for the first time and filled me with excitement for microbiology. Thanks for the countless hours you have invested in me, and for teaching me how to become a better scientist and teacher. This research was carried out at the Jet Propulsion Laboratory, California Institute of Technology, and was sponsored by the JPL Graduate Student Program and the National Aeronautics and Space Administration (80NM0018D0004). A special thanks to Arman Seuylemezian and Parag Vaishampayan, who taught me most of what I learned about lab work at JPL. It was a pleasure learning from you both, thank you for always being there when I needed your help. Thank you to Dr. Dixon and Dr. Vaishampayan for agreeing to be on my committee and all the work that it has entailed. A special thank you to my parents who supported me financially through my entire masters and bachelors’ program. Without your help, I never would have accomplished my goals without getting student loans. This has really meant a lot to me, and I can never thank you enough. A huge thank you to my girlfriend Raven, who has been with me through the entire program supporting me and keeping me motivated. iii Thanks to everyone else in the Lin lab for always being available to help. I feel like we all made the best of it together, even during these challenging times. Good luck to all of you! Finally, a big thanks to NASA and The Biotechnology and Planetary Protection Group at JPL, without your assistance and funding this research never would have been possible. iv ABSTRACT The spacecraft assembly clean rooms where spacecraft are assembled represent a unique microbiological ecosystem. Stringent bioburden reduction such as controlled temperature, humidity, HEPA filtered air circulation, regular decontamination, and gowning procedures are implemented throughout the assembly and testing operations. The microorganisms that survive these cleaning procedures show a unique microbial diversity and are a complex blend of human and soil associated flora that are often spore-forming. This taxonomic characterization study is essential for understanding which microorganisms were present on the surface of the Phoenix Mars Lander after microbial reduction measures. Knowing the bacterial contaminants' identities and whether they form spores helps NASA to predict whether the microorganisms pose a forward contamination risk that could affect future planetary protection policy. A combination of MALDI-TOF MS and 16S rRNA sequencing was used to identify bacterial isolates collected from the Phoenix Mars Lander. There was a total of 637 isolates analyzed using MALDI-TOF MS, approximately 73% (320) of the 438 microorganisms identified to belong to spore-forming genera. For the samples that could not be identified using MALDI-TOF MS method, the 16S rRNA sequencing approach was implemented. Of the 405 isolates identified with 16S rRNA, there were 312 (77%) isolates that were identified as spore-forming genera. After combining the results from these two methods there were 626 isolates identified, with 460 (74%) of the isolates identified as a spore- forming genus. This bias for spore-forming isolates could be due to many of the samples being heat-shocked at 80◦C for 15 minutes when using the NASA standard assay (NSA) for bioburden detection. v To better understand the relationship between the sample source and the prevalence of spore-forming bacteria, the unique microbiomes of three different locations on the Phoenix Mars Lander were compared. The three components analyzed were the Phoenix Mars Lander’s Fairing, MECA (Microscopy, Electrochemistry, and Conductivity Analyzer), and Robotic Arm. Since some of the sensitive electronics found on the Phoenix Mars Lander’s MECA and Robotic Arm have different sterilization procedures they could have a different frequency of spore-forming bacteria. A chi-square test of independence was performed to examine the relationship between the sample source and the presence of spore-forming bacteria. The relationship between these variables was found to be significant, and there have been fewer spore-forming isolates identified from the Fairing, MECA, and Robotic Arm, compared to the other locations. Further understanding of these complex interactions can provide insight for future Planetary Protection missions. vi TABLE OF CONTENTS SIGNATURE PAGE ......................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................ iii ABSTRACT ....................................................................................................................... v LIST OF TABLES ........................................................................................................... xi LIST OF FIGURES ........................................................................................................ xii 1. INTRODUCTION ..................................................................................................... 1 A. A brief introduction of the Jet Propulsion Laboratory ................................. 1 B. Phoenix Mars Lander ......................................................................................... 1 i. History of the Phoenix Mars Lander ............................................................. 1 ii. Objectives of the Phoenix Mars Lander ....................................................... 2 iii. Accomplishments of the Phoenix Mars Lander ......................................... 3 iv. Construction of the Phoenix Mars Lander ................................................. 4 C. The Planetary Protection Mission of JPL ........................................................ 6 i. The mission of COSPAR and Planetary Protection ..................................... 6 ii. The NASA Standard Spore Assay ................................................................ 7 iii. Current JPL Planetary Protection microbial collection ............................ 8 iv. Present and future research with GESAM ................................................. 9 D. Bacterial taxonomic identification using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) ......................... 9 i. History of MALDI-TOF MS........................................................................... 9 vii ii. MALDI-TOF MS matrix associated sample preparation ........................ 11 iii. MALDI-TOF MS Laser Desorption and Ionization ................................ 12 iv. MALDI-TOF MS Time of Flight ............................................................... 13 v. JPL in-house MALDI-TOF MS microbial database ................................. 14 vi. Comparison of MALDI-TOF MS proteomic fingerprint to JPL database ................................................................................................................................... 15 vii. Advantages of MALDI-TOF MS over 16S rRNA gene sequencing ...... 17 2. OBJECTIVE ............................................................................................................ 19 3. MATERIALS AND METHODS ............................................................................ 20 A. Bacterial sample collection from Phoenix Mars Lander .................................. 20 B. Revive isolates from JPL Planetary Protection microbial collection .............. 22 C. Rapid and systematic identification using MALDI-TOF MS ..................... 22 i. Direct transfer technique for MALDI-TOF MS ......................................... 23 ii. Direct transfer procedure ............................................................................ 23 iii. Tube extraction technique for MALDI-TOF MS..................................... 24 iv. Tube extraction procedure ......................................................................... 25 v. Taxonomic identification using Bruker MALDI Biotyper System .......... 27 D. Zymo DNA extraction of Phoenix Isolates .................................................... 27 E. Bacterial 16S rRNA gene amplification and sequencing ............................. 28 F. Comparison of rRNA gene sequences to the EZBioCloud and the SILVA LTP type strain databases .......................................................................................... 30 viii 4. RESULTS ................................................................................................................... 32 A. Retrieval of bacterial isolates from JPL culture collection freezers and the creation of pure cultures ............................................................................................
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