Restriction Endonucleases
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Regulation of ATP Levels in Escherichia Coli Using CRISPR
Tao et al. Microb Cell Fact (2018) 17:147 https://doi.org/10.1186/s12934-018-0995-7 Microbial Cell Factories RESEARCH Open Access Regulation of ATP levels in Escherichia coli using CRISPR interference for enhanced pinocembrin production Sha Tao, Ying Qian, Xin Wang, Weijia Cao, Weichao Ma, Kequan Chen* and Pingkai Ouyang Abstract Background: Microbial biosynthesis of natural products holds promise for preclinical studies and treating diseases. For instance, pinocembrin is a natural favonoid with important pharmacologic characteristics and is widely used in preclinical studies. However, high yield of natural products production is often limited by the intracellular cofactor level, including adenosine triphosphate (ATP). To address this challenge, tailored modifcation of ATP concentration in Escherichia coli was applied in efcient pinocembrin production. Results: In the present study, a clustered regularly interspaced short palindromic repeats (CRISPR) interference sys- tem was performed for screening several ATP-related candidate genes, where metK and proB showed its potential to improve ATP level and increased pinocembrin production. Subsequently, the repression efciency of metK and proB were optimized to achieve the appropriate levels of ATP and enhancing the pinocembrin production, which allowed the pinocembrin titer increased to 102.02 mg/L. Coupled with the malonyl-CoA engineering and optimization of culture and induction condition, a fnal pinocembrin titer of 165.31 mg/L was achieved, which is 10.2-fold higher than control strains. Conclusions: Our results introduce a strategy to approach the efcient biosynthesis of pinocembrin via ATP level strengthen using CRISPR interference. Furthermore coupled with the malonyl-CoA engineering and induction condi- tion have been optimized for pinocembrin production. -
Molecular Cloning of DNA Fragments Produced by Restriction
Proc. Natl. Acad. Sci. USA Vol. 73, No. 5, pp. 1537-1541, May 1976 Biochemistry Molecular cloning of DNA fragments produced by restriction endonucleases SailI and BamI (DNA joining/plasmid/insertional inactivation of genes/Drosophila melanogaster) DEAN H. HAMER AND CHARLES A. THOMAS, JR. Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115 Communicated by Bernard D. Dats, February 25,1976 ABSTRACT The highly specific restriction endonucleases cleaves various DNAs about once every 8 kb (kilobases), as SalI and BamI produce DNA fragments with complementary, compared to about once every 4 kb for EcoRI (16, 18). The cohesive termini that can be covalently joined by DNA ligase. resulting fragments have cohesive termini, and can be joined The Escherichia coli kanamycin resistance factor pML21 has to one another in head-to-tail, head-to-head, and one Sall site, at which DNA can be inserted without interfering probably with the expression of drug resistance or replication of the tail-to-tail orientation. Another highly specific restriction en- plasmid. A more convenient cloning vehicle can be made with donuclease that produces cohesive termini is BamI, from Ba- the tetracycline resistance factor pSC101, since insertion of cillus amyloliqefaciens H (G. Wilson and F. Young, personal DNA either at its single site for Sall or at that for BamI inacti- communication). We have shown that it cleaves D. melano- vates plasmid-specified drug resistance but not replication. To gaster DNA about once every 6 kb. This report describes the take advantage of this insertional inactivation, pSC101 was construction and verification of plasmid vehicles that allow one joined to a ColEl-ampicillin resistance plasmid having no Sall site, and to a ColEl-kanamycin resistance plasmid having no to clone and amplify potentially any DNA fragment produced BamI site. -
Gibson Assembly Cloning Guide, Second Edition
Gibson Assembly® CLONING GUIDE 2ND EDITION RESTRICTION DIGESTFREE, SEAMLESS CLONING Applications, tools, and protocols for the Gibson Assembly® method: • Single Insert • Multiple Inserts • Site-Directed Mutagenesis #DNAMYWAY sgidna.com/gibson-assembly Foreword Contents Foreword The Gibson Assembly method has been an integral part of our work at Synthetic Genomics, Inc. and the J. Craig Venter Institute (JCVI) for nearly a decade, enabling us to synthesize a complete bacterial genome in 2008, create the first synthetic cell in 2010, and generate a minimal bacterial genome in 2016. These studies form the framework for basic research in understanding the fundamental principles of cellular function and the precise function of essential genes. Additionally, synthetic cells can potentially be harnessed for commercial applications which could offer great benefits to society through the renewable and sustainable production of therapeutics, biofuels, and biobased textiles. In 2004, JCVI had embarked on a quest to synthesize genome-sized DNA and needed to develop the tools to make this possible. When I first learned that JCVI was attempting to create a synthetic cell, I truly understood the significance and reached out to Hamilton (Ham) Smith, who leads the Synthetic Biology Group at JCVI. I joined Ham’s team as a postdoctoral fellow and the development of Gibson Assembly began as I started investigating methods that would allow overlapping DNA fragments to be assembled toward the goal of generating genome- sized DNA. Over time, we had multiple methods in place for assembling DNA molecules by in vitro recombination, including the method that would later come to be known as Gibson Assembly. -
Supplementary Table S1. Table 1. List of Bacterial Strains Used in This Study Suppl
Supplementary Material Supplementary Tables: Supplementary Table S1. Table 1. List of bacterial strains used in this study Supplementary Table S2. List of plasmids used in this study Supplementary Table 3. List of primers used for mutagenesis of P. intermedia Supplementary Table 4. List of primers used for qRT-PCR analysis in P. intermedia Supplementary Table 5. List of the most highly upregulated genes in P. intermedia OxyR mutant Supplementary Table 6. List of the most highly downregulated genes in P. intermedia OxyR mutant Supplementary Table 7. List of the most highly upregulated genes in P. intermedia grown in iron-deplete conditions Supplementary Table 8. List of the most highly downregulated genes in P. intermedia grown in iron-deplete conditions Supplementary Figures: Supplementary Figure 1. Comparison of the genomic loci encoding OxyR in Prevotella species. Supplementary Figure 2. Distribution of SOD and glutathione peroxidase genes within the genus Prevotella. Supplementary Table S1. Bacterial strains Strain Description Source or reference P. intermedia V3147 Wild type OMA14 isolated from the (1) periodontal pocket of a Japanese patient with periodontitis V3203 OMA14 PIOMA14_I_0073(oxyR)::ermF This study E. coli XL-1 Blue Host strain for cloning Stratagene S17-1 RP-4-2-Tc::Mu aph::Tn7 recA, Smr (2) 1 Supplementary Table S2. Plasmids Plasmid Relevant property Source or reference pUC118 Takara pBSSK pNDR-Dual Clonetech pTCB Apr Tcr, E. coli-Bacteroides shuttle vector (3) plasmid pKD954 Contains the Porpyromonas gulae catalase (4) -
How to Clone Ref V1-2
A primer on cloning in E. coli Michael Nonet Department of Neuroscience Washington University Medical School St Louis, MO 63110 Draft 1.2.1 Nov. 10, 2019 Things to add: gel electrophoresis methods basic bacteriology methods Index Overview E. coli vectors! 6 Types of E. coli vectors! 6 Components of typical plasmid vectors! 6 Origin of replication! 6 Antibiotic resistance genes! 7 Master Cloning Sites! 7 LacZ"! 7 ccdB ! 8 Design of DNA constructs! 9 Choice of vector! 9 Source of insert! 9 Plasmids! 9 Genomic or cDNA! 9 Synthetic DNA! 10 Overview of different approaches to creating clones! 10 Restriction enzyme based cloning! 10 Single vs. double cut method! 11 Blunt vs “sticky” restriction sites! 11 Typical methodology for restriction enzyme cloning! 11 Golden Gate style cloning! 12 Typical methodology for Golden Gate cloning! 12 Gateway cloning! 13 Typical methodology for Gateway cloning! 14 Gibson assembly cloning! 15 Typical procedure for Gibson assembly! 15 Site directed mutagenesis! 16 PCR overlap approach! 16 DpnI mediated site directed mutagenesis! 16 Typical procedure for site directed mutagenesis! 17 Detailed methodology for all five methods Restriction enzyme cloning! 18 Designing the cloning strategy ! 18 Determining which vector to use! 18 Designing the insert fragment! 19 Designing primers! 19 Vector preparation! 19 Insert preparation! 19 Detailed protocol! 20 Step 1: Design oligonucleotides to amplify the product of interest! 20 Step 2: PCR amplification of insert DNA! 20 Step 3: Clean-up purification of PCR product! 21 Step 4: Digestion of products! 21 Step 5: Gel purification of products! 22 Step 6: Ligation! 22 Step 7: Transformation of E. -
The Cloning Guide the First Step Towards a Succesful Igem Project Igem Bonn NRP-UEA Igem
The Cloning Guide The first step towards a succesful iGEM Project iGEM Bonn NRP-UEA iGEM iGEM Manchester-Graz iGEM Evry iGEM Vanderbilt iGEM Minnesota Carnegie Mellon iGEM iGEM Sydney UIUC iGEM iGEM York iGEM Toulouse iGEM Pasteur UCLA iGEM iGEM Stockholm Preface The cloning guide which is lying before you or on your desktop is a document brought to you by iGEM TU Eindhoven in collaboration with numerous iGEM (International Genetically Engi- neered Machine) teams during iGEM 2015. An important part in the iGEM competition is the collabration of your own team with other teams. These collaborations are fully in line with the dedications of the iGEM Foundation on education and competition, the advancement of syn- thetic biology, and the development of an open community and collaboration. Collaborations between groups of (undergrad and overgrad) students can lead to nice products, as we have tried to provide one for you in 2015. In order to compile a cloning guide, several iGEM teams from all over the world have been con- tacted to cooperate on this. Finally fifiteen teams contributed in a fantastic way and a cloning guide consisting of the basics about nine different cloning methods and experiences of teams working with them has been realized. Without the help of all collaborating teams this guide could never have been realized, so we will thank all teams in advance. This guide may be of great help when new iGEM teams (edition 2016 and later) are at the point of designing their project. How to assemble the construct for you project, is an important choice which is possibly somewhat easier to make after reading this guide. -
Psp73 Vector Technical Bulletin #TB041
tb041.0906.qxp 9/25/2006 10:44 AM Page a Technical Bulletin pSP73 Vector INSTRUCTIONS FOR USE OF PRODUCT P2221. PRINTED IN USA. Revised 9/06 Part# TB041 AF9TB041 0906TB041 tb041.0906.qxp 9/25/2006 10:46 AM Page 1 pSP73 Vector All technical literature is available on the Internet at: www.promega.com/tbs/ Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on use of this system. E-mail: [email protected] I. Description..........................................................................................................1 II. Product Components and Storage Conditions ............................................1 III. pSP73 Vector Multiple Cloning Region and Circle Map..........................2 IV. pSP73 Vector Restriction Sites........................................................................4 V. Related Products ................................................................................................6 VI. Reference .............................................................................................................7 I. Description The pSP73 Vector (1) offers a wide range of restriction sites, providing greater versatility in cloning and transcription of RNA in vitro. The pSP73 Vector contains the SP6 and T7 RNA polymerase promoters and a unique multiple cloning region, which includes restriction sites for BglII, EcoRV, ClaI, EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, AccI, SalI, PstI, SphI, HindIII, PvuII and XhoI. The sequences of Promega vectors are available online at www.promega.com/vectors/ and are also available from the GenBank® database. II. Product Components and Storage Conditions Product Size Cat.# pSP73 Vector 20µg P2221 Storage Conditions: Store the pSP73 Vector at –20°C. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. -
Method for Cloning and Producing the Bglii Restriction Endonuclease
Europäisches Patentamt (19) European Patent Office Office européen des brevets (11) EP 0 619 373 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int. Cl.7: C12N 15/55, C12N 15/54, of the grant of the patent: C12N 1/21, C12P 21/00 24.05.2000 Bulletin 2000/21 (21) Application number: 94302258.2 (22) Date of filing: 29.03.1994 (54) Method for cloning and producing the BglII restriction endonuclease and modification methylase Verfahren zur Klonierung und zur Herstellung der Restriktionsendonuklease und Modifikationsmethylase BglII Procédé de clonage et de production de l'endonucléase de restriction BglII et de la méthylase correspondante (84) Designated Contracting States: (74) Representative: DE FR GB Davies, Jonathan Mark Reddie & Grose (30) Priority: 09.04.1993 US 17664 16 Theobalds Road London WC1X 8PL (GB) (43) Date of publication of application: 12.10.1994 Bulletin 1994/41 (56) References cited: EP-A- 0 248 678 (73) Proprietor: NEW ENGLAND BIOLABS, INC. • J. BACTERIOL., vol. 134, 1978 pages 338-344, Beverly Massachusetts 01915 (US) C.H. DUNCAN ET AL.; 'Biochemical and genetic properties of site-specific restriction (72) Inventors: endonucleases in Bacillus globigii' • Brooks, Joan E. • EUR. J. BIOCHEM., vol. 117, 1981 pages 395-399, Beverly, Massachusetts 01915 (US) R. IMBER AND T.A. BICKLE; 'Purification and • Heiter, Daniel F. properties of the restriction endonuclease BglII Groveland, Massachusetts 01834 (US) from Bacillus globigii' • Anton, Brian P. Boston MA 02116-1203 (US) Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. -
Restriction Endonucleases
Restriction Endonucleases TECHNICAL GUIDE UPDATE 2017/18 be INSPIRED drive DISCOVERY stay GENUINE RESTRICTION ENZYMES FROM NEB Cut Smarter with Restriction Enzymes from NEB® Looking to bring CONVENIENCE to your workflow? Simplify Reaction Setup and Double Activity of DNA Modifying Enzymes in CutSmart Buffer: Digestion with CutSmart® Buffer Clone Smarter! Activity Enzyme Required Supplements Over 210 restriction enzymes are 100% active in a single buffer, in CutSmart Phosphatases: CutSmart Buffer, making it significantly easier to set up your Alkaline Phosphatase (CIP) + + + double digest reactions. Since CutSmart Buffer includes BSA, there Antarctic Phosphatase + + + Requires Zn2+ Quick CIP + + + are fewer tubes and pipetting steps to worry about. Additionally, Shrimp Alkaline Phosphatase (rSAP) + + + many DNA modifying enzymes are 100% active in CutSmart Ligases: T4 DNA Ligase + + + Requires ATP Buffer, eliminating the need for subsequent purification. E. coli DNA Ligase + + + Requires NAD T3 DNA Ligase + + + Requires ATP + PEG For more information, visit www.NEBCutSmart.com T7 DNA Ligase + + + Requires ATP + PEG Polymerases: T4 DNA Polymerase + + + DNA Polymerase I, Large (Klenow) Frag. + + + DNA Polymerase I + + + DNA Polymerase Klenow Exo– + + + Bst DNA Polymerase + + + ™ phi29 DNA Polymerase + + + Speed up Digestions with Time-Saver T7 DNA Polymerase (unmodified) + + + Qualified Restriction Enzymes Transferases/Kinases: T4 Polynucleotide Kinase + + + Requires ATP + DTT T4 PNK (3´ phosphatase minus) + + + Requires ATP + DTT > 190 of our restriction enzymes are able to digest DNA in CpG Methyltransferase (M. SssI) + + + 5–15 minutes, and can safely be used overnight with no loss of GpC Methyltransferase (M. CviPI) + Requires DTT T4 Phage β-glucosyltransferase + + + sample. For added convenience and flexibility, most of these are Nucleases, other: supplied with CutSmart Buffer. -
A Short History of the Restriction Enzymes Wil A
Published online 18 October 2013 Nucleic Acids Research, 2014, Vol. 42, No. 1 3–19 doi:10.1093/nar/gkt990 NAR Breakthrough Article SURVEY AND SUMMARY Highlights of the DNA cutters: a short history of the restriction enzymes Wil A. M. Loenen1,*, David T. F. Dryden2,*, Elisabeth A. Raleigh3,*, Geoffrey G. Wilson3,* and Noreen E. Murrayy 1Leiden University Medical Center, Leiden, the Netherlands, 2EaStChemSchool of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, UK and 3New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA Received August 14, 2013; Revised September 24, 2013; Accepted October 2, 2013 ABSTRACT Type II REases represent the largest group of characterized enzymes owing to their usefulness as tools for recombinant In the early 1950’s, ‘host-controlled variation in DNA technology, and they have been studied extensively. bacterial viruses’ was reported as a non-hereditary Over 300 Type II REases, with >200 different sequence- phenomenon: one cycle of viral growth on certain specificities, are commercially available. Far fewer Type I, bacterial hosts affected the ability of progeny virus III and IV enzymes have been characterized, but putative to grow on other hosts by either restricting or examples are being identified daily through bioinformatic enlarging their host range. Unlike mutation, this analysis of sequenced genomes (Table 1). change was reversible, and one cycle of growth in Here we present a non-specialists perspective on import- the previous host returned the virus to its original ant events in the discovery and understanding of REases. form. These simple observations heralded the dis- Studies of these enzymes have generated a wealth of covery of the endonuclease and methyltransferase information regarding DNA–protein interactions and catalysis, protein family relationships, control of restric- activities of what are now termed Type I, II, III and tion activity and plasticity of protein domains, as well IV DNA restriction-modification systems. -
Promiscuous Restriction Is a Cellular Defense Strategy That Confers Fitness
Promiscuous restriction is a cellular defense strategy PNAS PLUS that confers fitness advantage to bacteria Kommireddy Vasua, Easa Nagamalleswaria, and Valakunja Nagarajaa,b,1 aDepartment of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India; and bJawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India Edited by Werner Arber, der Universitat Basel, Basel, Switzerland, and approved March 20, 2012 (received for review November 22, 2011) Most bacterial genomes harbor restriction–modification systems, ognition and cofactor utilization, whereas the cognate MTase is encoding a REase and its cognate MTase. On attack by a foreign very site-specific (14). In addition to its recognition sequence, DNA, the REase recognizes it as nonself and subjects it to restric- GGTACC, the enzyme binds and cleaves a number of noncanoni- tion. Should REases be highly specific for targeting the invading cal sequences (i.e., TGTACC, GTTACC, GATACC, GGAACC, foreign DNA? It is often considered to be the case. However, when GGTCCC, GGTATC, GGTACG, GGTACT), with the binding bacteria harboring a promiscuous or high-fidelity variant of the affinity ranging from 8 to 35 nM (14, 15). Accordingly, many of fi REase were challenged with bacteriophages, tness was maximal these sites are cleaved efficiently with kcat values varying from 0.06 − − under conditions of catalytic promiscuity. We also delineate pos- to 0.15 min 1 vs. 4.3 min 1 for canonical sites (15). The promis- sible mechanisms by which the REase recognizes the chromosome cuous activity of the enzyme is directed by a number of cofactors. as self at the noncanonical sites, thereby preventing lethal dsDNA In the presence of the physiological levels of Mg2+, the most breaks. -
NEB Golden Gate Assembly Kit (Bsai-Hfv2) E1601 Manual
INSTRUCTION MANUAL NEB® Golden Gate Assembly Kit (BsaI-HF®v2) NEB #E1601S/L 20/100 reactions Version 2.0_2/20 Table of Contents Golden Gate Assembly Overview ............................................................................................................................................................... 2 pGGAselect Destination Plasmid Description ............................................................................................................................................ 3 Insert Considerations ................................................................................................................................................................................... 4 Golden Gate Assembly Transformation and Plating Protocols ................................................................................................................... 5 Screening Protocols ..................................................................................................................................................................................... 6 Frequently Asked Questions (FAQs) .......................................................................................................................................................... 6 Golden Gate Assembly Tips ........................................................................................................................................................................ 8 Specifications ..............................................................................................................................................................................................