Plasmids 101 4th Edition PDF

Addgene's eBook Collection 101 Plasmids July 2023 4th Edition Plasmids 101 4th Edition 1 Addgene's eBook Collection Available as part of our 101 collection. Plasmids 101 Viral Vectors 101 Fluorescent Proteins 101 CRISPR 101 Science Career Guide Find us...

Addgene's eBook Collection 101 Plasmids July 2023 4th Edition Plasmids 101 4th Edition 1 Addgene's eBook Collection Available as part of our 101 collection. Plasmids 101 Viral Vectors 101 Fluorescent Proteins 101 CRISPR 101 Science Career Guide Find us Addgene.org blog.addgene.org facebook.com/addgene twitter.com/addgene instragram.com/addgene youtube.com/@addgene linkedin.com/company/addgene Plasmids 101 4th Edition 2 INTRODUCTION TO PLASMIDS 101 A By the Addgene Team ny newcomer who joins a molecular biology lab will undoubtedly be asked to design, modify, or construct a plasmid. Although the newcomer likely knows that a plasmid is a small circular piece of DNA often found in bacterial cells, additional guidance may be required to understand the specific components that make up a plasmid and why each is important. Our mission with this eBook, Plasmids 101, is to curate a onestop reference guide for plasmids. This resource is designed to educate all levels of scientists and plasmid lovers. It serves as an introduction to plasmids, allowing you to spend less time researching basic plasmid features and more time developing the clever experiments and innovative solutions necessary for advancing your field. Plasmids 101 4th Edition 3 CONTENTS Introduction to Plasmids 101 3 Chapter 1 What is a Plasmid? 8 A Brief History of Plasmids 9 What is a Plasmid? 12 Origin of Replication 14 The Promoter Region 17 Terminators and PolyA Signals 24 Methylation and Restriction Enzymes 28 Chapter 2 Bacteria 32 E. coli Strains for Protein Expression 33 Common Lab E. coli Strains 38 Environmental Plasmids 42 Transformation, Transduction, Conjugation, and Transfection 44 Chapter 3 Screening and Selecting 48 Antibiotic Resistance Genes 49 Blue-White Screening 52 Positive and Negative Selection 55 Colony PCR 57 Plasmids 101 4th Edition 4 Chapter 4 Controlling Expression 62 Protein Expression 63 Inducible Promoters 67 Repressible Promoters 71 Chapter 5 Common Cloning Techniques 78 Five Factors to Help You Choose the Right Cloning Method 79 Restriction Cloning 83 Golden Gate Cloning 90 TOPO Cloning 94 Sequence and Ligation Independent Cloning (SLIC) 97 CcdB - The Toxic Key to Efficient Cloning 100 Gateway Cloning 103 Gibson Assembly 110 Chapter 6 Eukaryotic Expression Vectors 116 Mammalian Vectors 117 Yeast Vectors 121 Multicistronic Vectors 125 Plasmids 101 4th Edition 5 CONTENTS Chapter 7 Viral Vector Elements 130 Viral Vectors – An Introduction 131 AAV: A Versatile Tool for Gene Expression in Mammals 134 Chapter 8 Plasmids That Glow 138 History of Fluorescent Proteins 139 Which Fluorescent Protein Should I Use? 140 Chapter 9 Plasmid Tags 144 Protein Tags 145 Tag Your Favorite Yeast Genes with Ease 150 Chapter 10 Genome Engineering 152 Introduction to Genome Engineering 153 Cre-Lox 156 Knockout / Knock-In Plasmids 160 Overview of TALEN Technology 165 FLEx Vectors 168 Sleeping Beauty Awakens for Genome Engineering 172 Plasmids 101 4th Edition 6 Chapter 11 Verifying your plasmid 174 How to Verify Your Plasmid 175 Using Snapgene 179 Six Tips for Analyzing and Troubleshooting DNA Sequencing Results 181 NGS Plasmid Quality Control at Addgene 183 Tips for Using BLAST to Verify Plasmids 186 Dimers and Multimers 193 Chapter 12 You’ve Made a Plasmid... Now What? 198 How to Name Your Plasmid 199 Plasmid Incompatibility 201 Codon Usage Bias 206 Optimizing Plasmids Yield 210 Control Plasmids 216 Chapter 13 Depositing Your Plasmids with Addgene 218 A Brief History of Addgene 219 Benefits of Depositing 220 The Deposit Spreadsheet 221 Acknowledgements and Final Words 224 Plasmids 101 4th Edition 7  CHAPTER 1 What is a Plasmid? Plasmids 101 4th Edition 8  CHAPTER 1 | WHAT IS A PLASMID? A Brief History of Plasmids B By Marcy Patrick | October 2015 ioblasts? Plasmagenes? In the 1940s and 50s, scientists were working to understand genetic cytoplasmic factors that could be transferred between cells. At the time, these extranuclear agents of heredity were thought of as everything from parasites, to symbionts, to genes and the labels applied to them were vague or contradictory, owing in part to the fact that very little was known about the role these factors played within an organism. SO HOW DID PLASMIDS GET THEIR NAME? In 1952, Joshua Lederberg set out to clarify the classification of these cytoplasmic inheritance factors. He proposed the catch-all term “plasmid” derived as a hybrid of “cytoplasm” and “id” (Latin for ‘it’), as “a generic term for any extrachromsomal hereditary determinant.” Plasmids 101 4th Edition 9 CHAPTER 1 | WHAT IS A PLASMID? His proposal, however, was basically ignored. A separate term, “episome”, defined as “a non- essential genetic element which could exist either autonomously or integrated into the chromosome” was proposed a few years later by Élie Jacob and François Wollman and became the widely adopted name for these elements. At the time, the use of episome seemed appropriate, especially since the Fertility, or F-factor discovered by Ester Lederberg in 1952 was noted to integrate into the E. coli chromosome in some cases. This terminology held until the 1960s when scientists began to study other extrachromosomal particles, particularly Resistance or R-factors. Like F-factors, R-factors could be transferred between bacteria via cell-to- cell contact; however, scientists noted that, unlike F-factors, the evidence did not support the idea that R-factors could integrate into the chromosome. the first plasmid “cloning” experiment. Dr. Cohen Thus the term “episome” was eventually dropped and colleagues treated a tetracycline resistant and we’ve been using “plasmid” ever since! plasmid, pSC101, and a newly developed kanamycin resistant plasmid, pSC102, with EcoRI and selected for E. coli transformants that were FROM NAPKINS TO NOTEBOOKS resistant to both. When this proved successful, pSC101 became the first plasmid cloning vector Although discovered in the early 1950s, it took until and molecular biology was never the same. the 1970s for plasmids to gain prominence in the scientific community. Prior to this, bacteriophage, Over the next few years genes from different especially lambda, was the tool of choice for bacterial (and eventually mammalian) species molecular biologists wanting to study bacterial were cloned into plasmids and new cloning vectors genetics. This all changed thanks, in part, to a such as pBR322, pACYC, and pUC were developed collaboration initiated at a Hawaiian deli in 1972. to provide higher copy number vectors that could Using a deli napkin for paper, a small group of be used in these cloning experiments. scientists including Stanley Falkow, Stanley Cohen, Herbert Boyer, and Charles Brinton concocted a Although plasmids started as a somewhat niche wild idea of using the newly discovered EcoRI area of research, they are now seen as a ubiquitous enzyme (and its predictable cut site) to develop tool that can be diversely applied to many different Plasmids 101 4th Edition 10  CHAPTER 1 | WHAT IS A PLASMID? experiments. Addgene was founded in order to molecular biology and have been key in advancing store, QC, curate, and distribute them all in the our knowledge in areas such as bacterial name of making it a little bit easier for scientists conjugation and recombination, replication and to conduct their research! Since their discovery in topology, and cloning and gene expression. n the 1950s, plasmids have impacted many areas of Further Reading CSHL Meeting: Plasmids: History & Biology EPISOME-MEDIATED TRANSFER OF DRUG RESISTANCE IN ENTEROBACTERIACEAE VIII.: Six-Drug Resistance R The Joshua Lederberg Papers Factor. Watanabe, Tsutomu, Chizuko Ogata, and Sachiko Sato. Journal of Bacteriology 88.4 (1964): 922–928. Joshua Lederberg’s Personal Perspective: Plasmid PubMed PMID: 14219055. (1952-1997) Transmissible Drug Resistance in an Epidemic DNA Cloning: A Personal View after 40 Years. Cohen, Strain of Salmonella Typhimurium. Datta, Naomi. Stanley N. Proceedings of the National Academy of The Journal of Hygiene 60.3 (1962): 301–310. Sciences of the United States of America. 110.39 (2013): PubMed PMID: 14025218. 15521–15529 PubMed PMID: 24043817. Construction of Biologically Functional Bacterial Cell Genetics and Hereditary Symbiosis Lederberg, Plasmids In Vitro. Cohen, Stanley N. et al. Joshua. Physiological Reviews 32.4 (1952) 403-430. Proceedings of the National Academy of Sciences of the United States of America 70.11 (1973): 3240–3244. Sex Compatibility in Escherichia Coli. Lederberg, PubMed PMID: 1422013. Joshua, Luigi L. Cavalli, and Esther M. Lederberg. Genetics 37.6 (1952): 720–730. PubMed PMID: 17247418. Uniform Nomenclature for Bacterial Plasmids: A Proposal. Novick, R P et al. Bacteriological Reviews 40.1 (1976): 168–189. PubMed PMID: 16350226. Plasmids 101 4th Edition 11 CHAPTER 1 | WHAT IS A PLASMID? What is a Plasmid? A By Margo R. Monroe and Marcy Patrick | April 2020 t their most basic level, plasmids are small circular pieces of DNA that replicate independently from the host’s chromosomal DNA. They are mainly found in bacteria, but also exist naturally in archaea and eukaryotes such as yeast and plants. In nature, plasmids provide one or more functional benefits to the host such as resistance to antibiotics, degradative functions, and/or virulence. All natural plasmids contain an origin of replication (which controls the host range and copy number of the plasmid) and typically include a gene that is advantageous for survival, such as an antibiotic resistance gene. Gateway, Gibson, etc). The cloning method is ultimately chosen based on the plasmid you In contrast, plasmids utilized in the lab are usually want to clone into. Regardless, once the cloning artificial and designed to introduce foreign DNA steps are complete, the vector containing the into another cell. Minimally, lab-created plasmids newly inserted gene is transformed into bacterial have an origin of replication, selection marker, and cells and selectively grown on antibiotic plates. cloning site. The ease of modifying plasmids and the ability of plasmids to self-replicate within a cell Importantly, because the bacteria from which make them attractive tools for the life scientist plasmids are isolated grow quickly and make more or bioengineer. of the plasmids as they grow, scientists can easily make large amounts of plasmid to manipulate and use in later work. HOW IS A PLASMID CONSTRUCTED IN THE LAB? HOW DO SCIENTISTS USE PLASMIDS? Due to their artificial nature, lab plasmids are commonly referred to as “vectors” or “constructs.” Generally, scientists use plasmids to manipulate To insert a gene of interest into a vector, scientists gene expression in target cells. Characteristics may utilize one of a variety of cloning methods such as flexibility, versatility, safety, and cost-ef- (restriction enzyme, ligation independent, fectiveness enable molecular biologists to broadly Plasmids 101 4th Edition 12  CHAPTER 1 | WHAT IS A PLASMID? utilize plasmids across a wide range of applica- Produce enzymes that will make specific, tions. Some common plasmid types include clon- controlled changes to an organism’s genome ing plasmids, expression plasmids, gene knock- (genome engineering) down plasmids, reporter plasmids, viral plasmids, and genome engineering plasmids. Produce synthetic viruses that can be used in research or for therapeutics Some of the many things that plasmids can be used to do include: Addgene has compiled various educational re- sources to facilitate plasmid use in the lab. Ad- Produce large amounts of a protein so that scien- dgene’s Molecular Biology Reference iincludes tists can purify and study it in a controlled setting information about molecular cloning, how to choose a plasmid vector, molecular biology tools Produce proteins that glow so that scientists and references, and how to maintain your plas- can track their location or quantity inside a cell mid stocks.. The guide also contains multiple protocols and troubleshooting tips to make plas- Monitor the level of a chemical in a particular mid usage as simple and straightforward as pos- environment sible. n Table 1.1 - Plasmid Vector Elements Vector Element Description DNA sequence that allows initiation of replication within a plasmid by recruiting Origin of Replication (ORI) transcriptional machinery proteins. Antibiotic Resistance Allows for selection of plasmid-containing bacteria. Multiple Cloning Site Short segment of DNA which contains several restriction sites allowing for the easy insertion (MCS) of DNA. In expression plasmids, the MCS is often downstream from a promoter. Insert Gene, promoter, or other DNA fragment cloned into the MCS for further study. Drives transcription of the target gene. Vital component for expression vectors: determines Promoter Region which cell types the gene is expressed in and the amount of recombinant protein produced. The antibiotic resistance gene allows for selection in bacteria. However, many plasmids also Selectable Marker have selectable markers for use in other cell types. A short single-stranded DNA sequence used as an initiation point for PCR amplification or Primer Binding Site sequencing. Primers can be exploited for sequence verification of plasmids. Plasmids 101 4th Edition 13 CHAPTER 1 | WHAT IS A PLASMID? Origin of Replication L By Kendall Morgan and Marcy Patrick | February 2014 et’s consider another basic element of will produce many plasmid copies and others any plasmid: the origin of replication produce just a few copies depending on how they or “replicon.” The replicon is comprised are regulated. Generally, control of replication is of the origin of replication (ORI) and all of its referred to as “relaxed” or “stringent” depending control elements. The ORI is the place where on whether the ORI is positively regulated by RNA DNA replication begins, enabling a plasmid to or proteins, respectively. A plasmid’s copy number reproduce itself as it must to survive within cells. has to do with the balance between positive and negative regulation and can be manipulated with The replicons of plasmids are generally mutations in the replicon. For example, the pMB1 different from those used to replicate the host’s ORI maintains about 20 copies per cell, while chromosomal DNA, but they still rely on the pUC – which differs by only two mutations – will host machinery to make additional copies. ORI produce as many as 700 copies per cell. sequences are generally high in As and Ts; A-T base pairs are held together with two hydrogen So, how do you choose? Ask yourself these bonds instead of three as G-C pairs are. As a result, questions: stretches of DNA that are rich in A-T pairs melt more readily at lower temperatures. When DNA Will the plasmid be used exclusively in E. melts, it gives the replication machinery room to coli? Gram negative bacteria in general? come in and get busy making copies. Both gram negatives and gram positives? Will you have only one plasmid type in your SO MANY ORIGINS, SO cells at a time? LITTLE TIME Do you want to make a lot of your plasmid? Is There are lots of ORIs out there so, for now, we’ve the gene toxic in high amounts? ignored those used in eukaryotic cells and viruses and focused only on those found in bacteria. Some It is always good to keep in mind that plasmids common ones you might see include ColE1, pMB1 with low to medium copy numbers can still express (which comes in a few slightly different but well massive amounts of protein given the proper known derivatives), pSC101, R6K, and 15A. Not promoter (stay tuned!) and growth conditions. all origins of replication are created equal. Some Plasmids 101 4th Edition 14  CHAPTER 1 | WHAT IS A PLASMID? Table 1.2 - Origin of Replication Common Vectors Copy Number* ORI Incompatibility Group Control pUC ~500-700 pMB1 (derivative) A Relaxed pBR322 ~15-20 pMB1 A Relaxed pET ~15-20 pBR322 A Relaxed pGEX ~15-20 pBR322 A Relaxed pColE1 ~15-20 ColE1 A Relaxed pR6K ~15-20 R6K** B Stringent pACYC ~10 P15A B Relaxed pSC101 ~5 pSC101 C Stringent pBluescript ~300-500 ColE1 (derivative) and F1*** A Relaxed pGEM ~300-500 pUC and F1 A Relaxed *Actual copy number varies.. **Requires pir gene for replication (reference). ***F1 is a phage-derived ORI that allows for the replication and packaging of ssDNA into phage particles. Plasmids with phage-derived ORIs are referred to as phagemids. Plasmids 101 4th Edition 15 CHAPTER 1 | WHAT IS A PLASMID? CHOOSING YOUR ORIGIN OF The insert REPLICATION WISELY Bacteria tend to maintain fewer copies of plasmids if they contain large inserts or genes that create a The best choice of ORI depends on how many toxic product. plasmid copies you want to maintain, which host or hosts you intend to use, and whether or not you The E. coli strain need to consider your plasmid’s compatibility with Most E. coli strains can be used to propagate one or more other plasmids. Generally speaking, plasmids, but end A- E. coli are best for high yields plasmids with the same ORIs are incompatible be- of plasmids. cause they will compete for the same machinery, creating an unstable and unpredictable environ- ment. As a rule, plasmids from the same group GROWTH CONDITIONS should not be co-transformed, so if you require two plasmids for an experiment, make sure they The amount of aeration, temperature, culture vol- have “compatible” ORIs. See the table below for ume, antibiotic, and medium can all affect copy more details. number. Some ORIs are temperature sensitive; other ORIs can be “tricked” into amplifying more Table 1.2 highlights common cloning vectors, their copies with the addition of Chloramphenicol – copy number, ORI, and incompatibility group. make sure your growth conditions aren’t working Note the A-C compatibility grouping is an arbitrary against you! designation, and plasmids from the same incom- patibility group should not be co-transformed.. THE CULTURE INOCULUM OTHER FACTORS THAT AFFECT Freshly streaked bacteria have higher copy num- COPY NUMBER bers – for optimal results always pick a single col- ony and do not subculture directly from glycerol Although the sequence and regulation of the ORI stocks, agar stabs, or liquid cultures. Incubation dramatically affect the copy number of a plasmid, for 12-16 hours tends to give higher copy num- other external factors contribute as well. These bers since the bacteria have just reached station- considerations are especially useful to keep ary phase, but the cells have not started to die. n in mind if you are planning to purify your plasmid DNA. Plasmids 101 4th Edition 16  CHAPTER 1 | WHAT IS A PLASMID? The Promoter Region N By Kendall Morgan, A. Max Juchheim, and Marcy Patrick | April 2014 ow that we can replicate our plasmid THE RNA POLYMERASES and make sure cells maintain it, the next step is getting the plasmid to RNA is transcribed from DNA using an RNA express our gene of interest. Enter the promoter - polymerase (RNAP). In bacteria, this is done by a the element responsible for initiating transcription single enzyme; however, eukaryotes have multiple of your insert. polymerases which are each responsible for a specific subset of RNAs. To gain this specificity, In practice, the term “promoter” describes the the eukaryotic RNAP can recognize and bind to combination of the promoter (RNA polymerase specific promoter elements. This means that the binding site) and operators (response elements). promoter present in your plasmid backbone must Promoters are about 100 to 1000 base pairs long be compatible with the type of RNA that needs to and found upstream of their target genes. The be made: if you want mRNA (for gene expression) sequence of the promoter region controls the you need to use an RNAP II promoter, whereas binding of the RNA polymerase and transcription small RNAs (such as shRNA) are transcribed from factors, therefore promoters play a large role the RNAP III promoters. This section features in determining where and when your gene of promoters for general RNAP II and RNAP III interest will be expressed. transcription; however, viral LTRs such as RNAP II ? Transcription factors binding to the promoter region (purple) of a gene (blue). Plasmids 101 4th Edition 17 CHAPTER 1 | WHAT IS A PLASMID? promoters are commonly employed in lentiviral To combat this, scientists have created synthetic and retroviral constructs. These are covered in promoters, which typically include some combina- our viral vectors resources. tion of other promoter elements, and tend to be more tightly regulated. PROMOTER SPECIFICITY COMMON PROMOTERS FOR Aside from choosing a promoter based on the EUKARYOTES AND PROKARYOTES type of RNA transcript, you will also need to make sure your plasmid has a promoter suited to work- Tables 1.3 and 1.4 list some of the most common ing in your host organism. Because transcription bacterial and mammalian promoters. These lists machinery differs between cell types or organ- are by no means exhaustive, but should be a good isms, promoters must be similarly variable. Bacte- place to start when trying to pick your perfect pro- rial promoters only work in prokaryotic cells and moter. n typically only in the same or closely related spe- cies from which they were derived. Similarly, the various eukaryotic cell types (mammalian, yeast, plants, etc.) require unique promoters and there is very little crossover. Generally speaking, pro- moters in bacteria are less diverse and complex, having fewer parts than those in eukaryotic cells. Some promoters are constitutively active and on all the time, while others are more carefully controlled. Regulated promoters might act only in certain tis- sues or at certain times in development or there may be ways to turn them on or off at will with a chemical, heat, or light. In the cell, promoters themselves are controlled by still other regulato- ry factors: enhancers, boundary elements, insu- lators, and silencers; however, some “leaky” tran- scription may occur. This is normally not a big issue for cells, but it may confound research results or even kill your cells if your gene of interest is toxic. Plasmids 101 4th Edition 18  CHAPTER 1 | WHAT IS A PLASMID? Table 1.3 - Eukarotyic Promoters Promoter Primarily RNA Description Expression Additional Considerations Used For Transcript Strong mammalian May contain an General expression promoter enhancer region. CMV mRNA Constitutive expression from the human Can be silenced in cytomegalovirus some cell types Strong mammalian Tends to give consistent General EF1a mRNA expression from human Constitutive expression regardless of expression elongation factor 1 alpha cell type or physiology Mammalian expression General May include an SV40 mRNA promoter from the Constitutive expression enhancer simian vacuolating Widespread expression, PGK1 but may vary by cell type. (human Mammalian promoter General Tends to resist promoter or mRNA phosphoglycerate kinase Constitutive expression down regulation due mouse) gene to methylation or deacetylation. Mammalian promoter General As the name implies, this Ubc mRNA from the human Constitutive expression promoter is ubiquitous. ubiquitin C gene Ubiquitous. Chicken human Mammalian promoter version is commonly General beta mRNA Constitutive expression from beta actin gene used in promoter actin hybrids Contains CMV enhancer, General Strong hybrid chicken beta actin CAG mRNA Constitutive expression mammalian promoter promoter, and rabbit be- ta-globin splice acceptor. Plasmids 101 4th Edition 19 CHAPTER 1 | WHAT IS A PLASMID? Table 1.3 - Eukarotyic Promoters Promoter Primarily RNA Description Expression Additional Considerations Used For Transcript Typically contains a minimal promoter with Inducible low basal activity and General Tetracycline response with several tetracycline TRE mRNA expression element promoter Tetracyline or operators. Transcription its derivatives can be turned on or off depending on what tet transactivator is used. Drosophila promoter Requires the presence General UAS mRNA containing Gal4 Specific of Gal4 gene to activate expression binding sites promoter. Strong insect promoter Commonly used in General Ac5 mRNA from Drosophila Actin 5c Constitutive expression systems for expression gene Drosophila Commonly used in Polyhedrin General Strong insect promoter expression systems for mRNA Constitutive expression from baculovirus insect cells Used for neuronal/CNS Ca2+/calmodulin- General expression. Modulated CaMKIIa mRNA dependent protein Specific expression by calcium and kinase II promoter calmodulin. Inducible with Can be used Adjacent, divergently General galactose; independently or GAL1, 10 mRNA transcribed promoters expression repressible together. Regulated by from yeast with glucose GAL4 and GAL 80. Plasmids 101 4th Edition 20  CHAPTER 1 | WHAT IS A PLASMID? Table 1.3 - Eukarotyic Promoters Promoter Primarily RNA Description Expression Additional Considerations Used For Transcript Yeast transcription Analogous to General TEF1 mRNA elongation factor Constitutive mammalian EF1a expression promoter promoter. Strong yeast promoter General from glyceraldehyde Very strong, also called GDS mRNA Specific expression 3-phosphage TDH3 or GAPDH. dehydrogenase Full length version is strong with high General Yeast promoter for Repressed by expression. Truncated ADH1 mRNA expression alcohol dehydrogenase I ethanol promoters are constitutive with lower expression. Strong plant promoter Active in dicots, less General from the Cauliflower active in monocots, with CaMV35S mRNA Constitutive expression Mosaic Virus some activity in animal cells. General Plant promoter from Gives high expression in Ubi mRNA Constitutive expression maize ubiquitin gene plants. Small RNA May have slightly lower From the human expression expression than U6. May H1 shRNA polymerase III RNA Constitutive have better expression promoter in neuronal cells. Small RNA Murine U6 is also expression From the human U6 U6 shRNA Constitutive used, but may be less small nuclear promoter efficient. Plasmids 101 4th Edition 21 CHAPTER 1 | WHAT IS A PLASMID? Table 1.4 - Prokaryotic Promoters Promoter Primarily Description Expression Additional Considerations Used For In vitro transcription/ Promoter from T7 Constitutive, but Can be used for in vitro transcription general bacteriophage requires T7 RNA only if 2 different phage promoters T7 expression polymerase. are present in opposite orientations to gene. Negligible basal expression when Commonly found in pET vectors. Promoter from T7 not induced. Very tightly regulated by the lac High levels of bacteriophage plus Requires T7 RNA operators. Good for modulating gene expression T7lac lac operators polymerase, which gene expression through varied is also controlled inducer concentrations. by lac operator. Can be induced by IPTG. In vitro transcription/ Promoter from Sp6 Can be used for in vitro transcription Constitutive, but general bacteriophage only if 2 different phage promoters Sp6 requires SP6 RNA expression are present in opposite orientations polymerase. to gene. High levels of Promoter from E. Gets turned off with high levels of trp gene expression coli tryptophan Repressible cellular tryptophan. operon Constitutive in Leaky promoter with somewhat the absense of lac weak expression. lacIq mutation repressor (lacI increases expression of the General Promoter from lac lac or lacIq). Can be repressor 10x, thus tightening expression operon induced by IPTG or regulation of lac promoter. Good for lactose. modulating gene expression through varied inducer concentrations. Contains -35 region from trpB Regulated like the and -10 region from lac. Very tight General Hybrid promoter lac promoter regulation. Good for modulating Ptac expression of lac and trp gene expression through varied inducer concentrations. Generally better expression than lac alone. Plasmids 101 4th Edition 22  CHAPTER 1 | WHAT IS A PLASMID? Table 1.4 - Prokaryotic Promoters Promoter Primarily Description Expression Additional Considerations Used For High levels of Promoter from Can be Often paired with the temperature pL gene expression bacteriophage temperature sensitive cI857 repressor. lambda regulatable Inducible by arabinose and repressed Weaker. Commonly found in pBAD Promoter of via catabolite vectors. Good for rapid regulation General the arabinose repression in the and low basal expression; expression araBAD metabolic operon presence however, not well-suited for of glucose or modulating gene expression through by competitive varied inducer concentrations. binding of the anti- inducer fucose Plasmids 101 4th Edition 23 CHAPTER 1 | WHAT IS A PLASMID? Terminators and Poly(a) Signals P By Julian Taylor-Parker | May 2016 l asmids designed to express genes post-transcriptional additional of multiple ad- in a given host cell type are generally enine (A) nucleotides to the tail of a messenger broken down into two broad categories, RNA transcript. The purpose and mechanism of prokaryotic or eukaryotic, based on the functional polyadenylation vary among cell types, but polya- elements they contain. Plasmid DNA in both denylation generally serves to promote transcript prokaryotic and eukaryotic systems must be longevity in eukaryotes and promote transcript transcribed into RNA. Transcription occurs in three degradation in prokaryotes. phases: initiation, elongation, and termination. We previously discussed the promoter’s role in the initiation step of gene transcription; here we’ll PROKARYOTIC TERMINATION provide an overview on how transcription stops, or termination. Prokaryotic termination mechanisms fall under two general categories: rho-dependent and rho- independent. Rho factor is a helicase which as- WHAT IS TERMINATION AND sists RNA polymerase in the termination of the POLYADENYLATION? transcript. Rho-dependent terminators are not usually employed in plasmid-based expression The role of the terminator, which is a se- systems, so these will not be detailed here, but quence-based element, is to define the end of a additional references are provided at the end. transcriptional unit (such as a gene) and initiate the process of releasing the newly synthesized RNA Nearly all common bacterial expression plasmids from the transcription machinery. Terminators are use Rho-independent terminators, which include found downstream of the gene to be transcribed, naturally occurring terminators, such as T7 and and typically occur directly after any 3’ regulatory rrnB, as well as engineered high-efficiency termi- elements, such as the polyadenylation or poly(A) nators such as T0. Rho-independent termination signal. While many studies focus on promoter is also known as intrinsic termination, and relies strength as a determinant of gene expression lev- on the formation of a GC-rich hairpin in the RNA els, the terminator also plays an important role in transcript followed by a weakly bound poly-ura- RNA processing and contributes to variability in cil tract as shown in the figure to the right. The RNA half-life, and ultimately gene expression. tertiary structure of the hairpin-DNA complex is thought to destabilize the transcription complex, Polyadenylation, as the name implies, is the initiating cleavage of the transcript. Plasmids 101 4th Edition 24  CHAPTER 1 | WHAT IS A PLASMID? No terminator is 100% efficient at halting transcription of the template and initiating the desired cleavage event, although some engineered terminators come close (>95%). For most purposes, however, any common terminator will suffice. Many commercial expression vectors use double terminators to reduce unwanted translation of downstream elements. A high affinity terminator may be desired for multicistronic constructs where high termination efficiency is necessary to minimize transcriptional read-through.. Chris Voigt’s lab has characterized a set of prokaryotic terminators and deposited several with Addgene. PROKARYOTIC POLYADENYLATION Although mostly thought of as a eukaryotic- specific process, prokaryotes also add poly(A) tails to certain RNAs. Unlike the eukaryotic mechanism A predicted conserved secondary structure and sequence which requires a consensus sequence for the conserved Rho-independent termination annotation for 90 addition of a poly(A) tail, the addition of a poly(A) bacterial elements. By Ppgardne at English Wikipedia, tail on a prokaryotic transcript is non-specific CC BY-SA 3.0 and can be added to any accessible 3’ end. The presence of the poly(A) tail targets the RNA to the degradosome, which contains enzymes that cut RNA not protected by secondary structure. Because it lacks specificity, it is thought that poly(A) s are used to control the cellular concentration of Plasmids 101 4th Edition 25  CHAPTER 1 | WHAT IS A PLASMID? regulatory RNAs and may additionally act as a Mammalian expression plasmids are primari- quality control mechanism to rid the cell of ly used to create mRNA and the commonly used misfolded RNAs. mammalian terminators (SV40, hGH, BGH, and rb- Glob) include the sequence motif AAUAAA which EUKARYOTIC TERMINATION AND promotes both polyadenylation and termination. POLYADENYLATION Out of those listed, the SV40 late polyA and rbGlob polyA are thought to be more efficient in terminat- Unlike prokaryotes that have a single RNA poly- ing transcription due to the presence of addition- merase for transcription, eukaryotes have three al helper sequences (Schek et. al., 1992; Gil et. al, RNA polymerases (Polymerases I, II, and III), each 1987). responsible for transcribing different types of RNA: Polymerase I is responsible for ribosomal As alluded to above, termination and polyade- RNA, Polymerase II is responsible for mRNA and nylation of Polymerase II transcripts (and there- miRNAs, and Polymerase III transcribes tRNA and fore mRNAs) are coordinated processes. Cleavage other short RNAs. Although not as well studied between the consensus motif and a downstream as prokaryotic termination, the basic processes GU-rich region (shown in the figure below) releas- for eukaryotic termination are understood and es the mRNA from the polymerase and creates a it has been noted that each eukaryotic RNA poly- free 3’ end which is now available for polyadenyla- merase terminates differently. Polymerase III, for tion. The addition of the poly(A) tail is important example, relies on a specific sequence and RNA for stability of the mRNA, protection from degre- secondary structure to induce transcript cleav- dation, and is integral to the nuclear export and age, similar to the Rho-independent termination translation processes as well. n found in prokaryotes. This is different than Poly- merases I and II, which both require binding of termination factors. Although both are termination factor dependent, Polymerases I and II employ different mechanisms to terminate transcription. Polymerase I uses a process similar to the prokaryotic Rho-depen- dent mechanism, whereas Polymerase II termina- tion is more complex and involves two RNA poly- merase-associated proteins, CPSF and CstF, which are responsible for recruiting the cleavage and polyadenylation enzymes, in a process that seems to couple termination with polyadenlyation. Plasmids 101 4th Edition 26  CHAPTER 1 | WHAT IS A PLASMID? Further Reading Peters JM, Vangeloff AD, Landick R. Bacterial Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck Transcription Terminators: The RNA 3′-End Hepatitis Virus Posttranscriptional Regulatory Chronicles. Journal of molecular biology. 2011. Element Enhances Expression of Transgenes PubMed PMID: 21439297. PubMed Central PMCID: Delivered by Retroviral Vectors. Journal of Virology. PMC3622210. 1999. PubMed PMID: 10074136. PubMed Central PMCID: PMC104046. Schek N, Cooke C, Alwine JC. Definition of the upstream efficiency element of the simian virus Wodrich H, Schambach A, Kräusslich H-G. 40 late polyadenylation signal by using in vitro Multiple copies of the Mason–Pfizer monkey analyses. Molecular and Cellular Biology. 1992. virus constitutive RNA transport element lead PubMed PMID: 1333042. PubMed Central PMCID: to enhanced HIV-1 Gag expression in a context- PMC360476.. dependent manner. Nucleic Acids Research. 2000. PubMed PMID:10648781. PubMed Central PMCID: Gil A, Proudfoot NJ. Position-dependent sequence PMC102582. elements downstream of AAUAAA are required for efficient rabbit beta-globin mRNA 3’ end formation. Cell. 1987. PubMed PMID: 3568131. Hager S, Frame FM, Collins AT, Burns JE, Maitland NJ. An Internal Polyadenylation Signal Substantially Increases Expression Levels of Lentivirus-Delivered Transgenes but Has the Potential to Reduce Viral Titer in a Promoter-Dependent Manner. Hum Gene Ther. 2008. PubMed PMID: 18627247. Plasmids 101 4th Edition 27 CHAPTER 1 | WHAT IS A PLASMID? Methylation and Restriction Enzymes H By Marcy Patrick | June 2016 ave you ever tried digesting with endonucleases that recognizes and cleave the XbaI or ClaI restriction enzymes same DNA sequence if not methylated (if it comes and gotten unusual or unexpected from a phage for instance). results? Or considered why DpnI will degrade your template DNA from a PCR reaction but not the Aside from restriction modification systems, newly synthesized product from a site-directed DNA methylation also plays an integral role mutagenesis experiment? The answer to both in regulating genome replication, repairing questions is the same - methylation! Read on to mismatched basepairs or small indels that learn about how DNA methylation may affect your occur during DNA synthesis, and protomoting restriction digests. or repressing protein expression. Methylases involved with these processes (for example Dam and Dcm methylases) are independent from the restriction modification systems, yet can still WHY METHYLATE? affect whether certain restriction enzymes can effectively cleave DNA. Aside from restriction modification systems, DNA methylation also plays an integral role Common lab E. coli K12 strains such as DH5alpha in regulating genome replication, repairing contain 3 methylases that recognize and methylate mismatched basepairs or small indels that different stretches of DNA: occur during DNA synthesis, and promoting or repressing protein expression. It turns out that Dam methylase adds a methyl group to the restriction enzymes are one half of naturally adenine of GATC stretches of DNA occuring restriction modification systems that prokaryotes use to protect themselves from Dcm methylase adds a methyl group to the foreign DNA. The other component of these second cytosine of CCWGG systems, methyltransferases, methylate DNA at particular sequences to prevent them from EcoKI methylase adds a methyl group being degraded by restriction endonucleases. to the adenine in AACNNNNNNGTGC or A given prokaryote typically has genes encoding GCACNNNNNNGTT one or a few restriction modification systems containing methyltransferases that add methyl groups to specific DNA sequences and companion Plasmids 101 4th Edition 28  CHAPTER 1 | WHAT IS A PLASMID? Figure 1 IMPACTS ON CLONING AND DIGESTS would either be difficult to interpret or lead you to conclude your plasmid is not correct! Although not all prokaryotic DNA is methylated to the same level, the potential for methylation Conversely, enzymes such as DpnI require should be considered when digesting DNA. Why? methylation at their recognition sites in order Well, even though Dam methylation sites are to efficiently cleave DNA. DpnI is often used for not specifically associated with any restriction site directed mutagenesis. During this process, modification systems, their sequences may incorporation of a desired mutation into your overlap with restriction sites, inhibiting enzymes plasmid of interest by PCR generates mutated such as ClaI or XbaI, or, conversely, activating plasmids with no methylation (there are no enzymes such as DpnI. methyltransferases in the PCR reaction). The template plasmid, on the other hand, should For example, cleavage by XbaI may be blocked be derived from a dam+ E. coli strain and will due to methylation if the enzyme’s recognition site therefore have methylated adenines in any GATC (TCTAGA) is preceded by GA or followed by TC. As sequences found in the plasmid. When the PCR shown in Figure 1, a Dam methylase recognition products are digested with DpnI, only the non- site (underlined in red) overlaps with the XbaI mutated and methylated template is destroyed cut site (depicted as an orange line) because leaving behind a pool of mutated plasmids which the restriction site is followed by TC.Addition can later be verified by Sanger sequencing. of the methyl group at this site would block the enzyme from cutting the DNA there, although other restriction sites (including other XbaI sites HOW CAN I TELL IF MY ENZYME not preceeded by GA or followed by TC) would WILL CUT? be cut as normal. If not taken into account, this blocked site could potentially give you results that Conversely, enzymes such as DpnI require Plasmids 101 4th Edition 29 CHAPTER 1 | WHAT IS A PLASMID? methylation at their recognition sites in order to efficiently cleave DNA. DpnI is often used for site directed mutagenesis. During this process, incorporation of a desired mutation into your plasmid of interest by PCR generates mutated plasmids with no methylation (there are no methyltransferases in the PCR reaction). The template plasmid, on the other hand, should be derived from a dam+ E. coli strain and will therefore have methylated adenines in any GATC sequences found in the plasmid. When the PCR products are digested with DpnI, only the non- Image created using Snapgene(R). mutated and methylated template is destroyed leaving behind a pool of mutated plasmids which can later be verified by Sanger sequencing. re-purifying it. These specialized E. coli strains have been specifically engineered to be Dam and Dcm methylase-deficient, and, as such, produce CONTROLLING METHYLATION DNA that is unmethylated at those sites. Please keep in mind that dam-/dcm- strains may have Whether performing a digest for cloning purposes an increased rate of mutation (as these would or for diagnostics, we suggest double checking also be deficient in mismatch repair functions to make sure your results will not be affected of Dam), so these strains should not be used for by methylation. Conveniently, the majority of long term storage. n restriction enzymes commonly used in the lab do not have recognition sites that could overlap with a methylation site. The quick-reference table below lists 10 common enzymes that may be affected by methylation. This table is by no means exhaustive, Further Reading so you may want to consult the REBASE database for more detailed information. Review of Restriction Modification Systems Finally, you can control methylation by altering NEB’s Resource Describing Dam and your choice of bacteria. For example, if you must Dcm Methylases use a restriction site that will be blocked by Dam or Dcm methylation, you can ensure this site Discovery of DpnI remains unblocked by first cloning your DNA into a dam–/dcm– strain of E. coli such as JM110 and Review describing dam-/dcm- E. coli Plasmids 101 4th Edition 30  CHAPTER 1 | WHAT IS A PLASMID? Table 1.5 - Dam Methylation Enzymes Enzyme Dam methylation Dcm methylation EcoKI methylation ApaI Not affected Blocked by overlapping methylation Not affected BsaI Not affected Blocked by overlapping methylation Not affected Blocked by overlapping ClaI Not affected Not affected methylation DraI Blocked by overlapping Not affected Not affected methylation Blocked by overlapping HpaI Not affected Not affected methylation Blocked by overlapping MboI Not affected Not affected methylation MboI Not affected Blocked by overlapping methylatio Not affected Blocked by overlapping PmeI Not affected Not affected methylation Blocked by overlapping XbaI Not affected Not affected methylation Requires methylation DpnI Not affected Not affected for activity Plasmids 101 4th Edition 31  CHAPTER 2 Bacteria Plasmids 101 4th Edition 32  CHAPTER 2 | BACTERIA E. Coli Strains for Protein Expression W By Julian Taylor-Parker | February 2015 e previously reviewed the salient features of several popular strains of E. coli for DNA propagation. While great for cloning purposes, these E. coli strains are not usually well suited for recombinant protein expression. Many challenges can arise when over-expressing a foreign protein in E. coli. We will review the potential pitfalls of recombinant protein expression and some of the most popular commercial strains designed to avoid them. Plasmids 101 4th Edition 33 CHAPTER 2 | BACTERIA WHY DO I NEED AN system. This means the strain can neither restrict nor methylate DNA. EXPRESSION STRAIN? Protein expression from high-copy number plasmids and powerful promoters will greatly HOW DOES INDUCIBLE exceed that of any native host protein, using up EXPRESSION WORK? valuable resources in the cell thus leading to slowed growth. Additionally, some protein products may As mentioned above, many expression plasmids be toxic to the host when expressed, particularly utilize inducible promoters, which are ‘inactive’ those that are insoluble, act on DNA, or are until an inducer such as IPTG is added to the enzymatically active. For this reason, recombinant growth medium. Induction timing is important, proteins are typically expressed in E. coli as you typically want to make sure your cells have engineered to accommodate high protein loads first reached an appropriate density. Cells in the using inducible promoter systems (which will be exponential growth phase are alive and healthy, discussed later). In addition to the basic genotypes which makes them ideal for protein expression. If outlined below, certain specialized strains are you wait too long to induce, your culture will start available to confer greater transcriptional control, collecting dead cells, and, conversely, you cannot assist with proper protein folding, and deal with induce too early as there are not enough cells in sub-optimal codon usage (Table 2.1). the culture to make protein. A few mutations are common to all or most The DE3 lysogen/T7 promoter combination is expression strains to accommodate high protein the most popular induction system. The DE3 levels including: lysogen expresses T7 RNA polymerase (RNAP) from the bacterial genome under control of the ompT: Strains harboring this mutation are lac repressor, which is inducible by the addition deficient in outer membrane protease VII, of IPTG. T7 RNAP is then available to transcribe which reduces proteolysis of the expressed the gene of interest from a T7 promoter on the recombinant proteins. plasmid. Many commercial strains carry the DE3 lysogen, as indicated by the name of the strain. lon protease: Strains where this is completely Conversely, other strains such as M15(pREP4) use deleted (designated lon or Δlon) similarly a lac repressor to act directly on the expression reduce proteolysis of the expressed proteins. plasmid in order to repress transcription from a hybrid promoter. hsdSB (rB- mB- ): These strains have an inactivated native restriction/methylation Although the DE3/T7 RNAP system works well for most experiments, the lac promoter can “leak,” Plasmids 101 4th Edition 34  CHAPTER 2 | BACTERIA Table 2.1 - E. coli Expression Strains Strain Resistance Key Features Genotype Use BL21(DE3) N/A Basic IPTG-inducible strain F- ompT lon hsdSB(rB- General protein containing T7 RNAP (DE3) mB-) gal dcm (DE3) expression BL21(DE3) Chloramphenicol pLysS expresses T7 lysozyme to F- ompT lon hsdSB(rB- Expression of (pLysS) reduce basal expression levels; mB-) gal dcm(DE3) toxic proteins expression vector cannot have pLysS (CamR) p15A origin of replication pLysS* Chloramphenicol pLysE has higher T7 lysozyme F- ompT lon hsdSB Expression of BL21(DE3) (pLysE) expression than pLysS; expression toxic proteins vector cannot have p15A origin of replicationRNAP (DE3) pLysE* (rB- mB-) gal dcm(DE3) pLysE (CamR) BL21 star N/A Lacks functional RNaseE which F- ompT lon hsdSB(rB- General (DE3) results in longer transcript half-life mB-) gal dcm rne131 expression; not (DE3) recommended for toxic proteins BL21-A1 Tetracycline Arabinose-inducible expression F- ompT lon hsdSB(rB- General protein of T7 RNAP; IPTG may still be mB-) gal dcm expression required for expression araB::T7RNAP-tetA BLR (DE3) Tetracycline RecA-deficient; best for plasmids F- ompT lon hsdSB(rB- Expression with repetitive sequences. mB-) gal dcm(DE3) of unstable Δ(srl- recA)306::Tn10 proteins (TetR) HMS174 Rifampicin RecA-deficient; allows for cloning F- recA1 hsdR(rK12- Expression (DE3)** and expression in same strain mK12+) (DE3) (RifR) of unstable methylation proteins Contains mutated lac permease Expression of Tuner F- ompT lon hsdSB(rB- N/A which allows for linear control of toxic or insoluble (DE3) mB-) gal dcm lacY1(DE3) expression proteins Plasmids 101 4th Edition 35 CHAPTER 2 | BACTERIA Table 2.1 - E. coli Expression Strains Strain Resistance Key Features Genotype Use Rosetta2 Chloramphenicol Good for “universal” translation; F- ompT hsdSB(rB- Expression of (DE3)* (pRARE) contains 7 additional tRNAs for mB-) gal dcm (DE3) eukaryotic proteins rare codons not normally used in pRARE2 (CamR) Lemo21 Chloramphenicol Rhamnose-tunable T7 RNAP fhuA2 [lon] ompT gal Expression of (DE3)* (pLemo) expression alleviates inclusion (λ DE3) [dcm] ΔhsdS/ toxic, insoluble, body formation. Expression pLemo (CamR) or membrane vector cannot have p15A origin proteins of replication T7 Chloramphenicol IPTG-inducible expression of T7 fhuA2 lacZ::T7 General protein (pRARE) RNAP from the genome; does not gene1 [lon] ompT expression restrict methylated DNA gal sulA11 R(mcr- 73::miniTn10- m15** Cis-repression of the E. coli T5 F-, Φ80ΔlacM15, thi, Expression of toxic pREP4* Kanamycin (pREP4) promoter (found on vectors such proteins as pQE or similar) inducible under IPTG (lac repressor on the pREP4 plasmid). Expression vector cannot have p15A origin of replication Origami2 Streptomycin and Contains highly active Δ(ara-leu)7697 Expression of (DE3)** Tetracycline thioredoxin reductase and ΔlacX74 ΔphoA PvuII insoluble proteins glutathione reductase to facilitate phoR araD139 ahpC proper folding; may increase galE galK rpsL F′[lac+ multimer formation lacIq pro] (DE3) gor522::Tn10 trxB (StrR, TetR) Note: All strains are derived from the E.coli B strain, except those marked with ** which are K12. * indicates that the strain component is a plasmid. Plasmids 101 4th Edition 36  CHAPTER 2 | BACTERIA meaning that a low level of expression exists example, nor will a p15 plasmid be compatible with even without the addition of IPTG. This is mostly a pLys strain. Your strain may require additional a problem for toxic protein products, which can antibiotic selection or a special growth media, or prevent the culture from reaching the desired if your plasmid is low-copy, consider reducing the density within a reasonable timeframe. For these antibiotic concentration. cases, some strains carry an additional measure of control such as the pLys plasmid, which Growth temperature suppresses basal T7 expression. The pLys plasmid contains a chloramphenicol resistance cassette for Analyze your expression conditions by setting up a positive selection and a p15A origin of replication, small-scale expression experiment to test variables making it incompatible with other p15A plasmids. such as temperature, time, and media conditions. pLys comes in two flavors—pLysS and pLysE—the Many recombinant proteins express better at 30 difference being that the latter provides tighter °C or room temperature, which is accomplished control of basal expression. by growing your culture to the desired density at 37 °C and reducing the temperature or moving it to a bench-top shaker 10—20 minutes before WHAT IF I DON’T SEE PROTEIN adding the inducer. OVEREXPRESSION? Growth media The strains described above should generate sufficient expression levels for most purposes, Changing media is tricky, because there can be a but what do you do when you’ve tried a common trade-off between growth rate and protein quality. strain and don’t get the desired level (or any) For many proteins, a rich media such as TB or 2XYT protein expression? Low expression outcomes is optimal because of the high cell-density they can result from variety of sources, so fear not— support; however, minimal media supplemented there are a few simple troubleshooting measures with M9 salts may be preferable if the protein that can help get you back on track: product is secreted to the medium or if slow expression is required due to solubility concerns. Compatibility Insoluble and secreted proteins Double-check your plasmid backbone and expression strain to make sure they are The most common purification protocols are compatible. An arabinose-inducible plasmid designed for soluble, cytosolic protein products, will not express in an IPTG induction strain for but this is not always achievable. Proteins Plasmids 101 4th Edition 37 CHAPTER 2 | BACTERIA which contain hydrophobic regions or multiple disulfide bonds may aggregate and become insoluble. These insoluble globs of misfolded protein are known as inclusion bodies and can be recovered and purified using a special protocol. Alternatively, reducing the concentration of inducer or adding an affinity tag such as GST may help with solubility issues. n Plasmids 101 4th Edition 38  CHAPTER 2 | BACTERIA Common Lab E. coli Strains Y By Matthew Ferenc | November 2014 ou’ve worked hard designing your are mainly found in the intestinal tract of animals. plasmid – you carefully selected the There are many different naturally occurring optimal promoter for your gene strains of E. coli, some of which are deadly to of interest, you painstakingly cloned into the humans. The majority of all common, commercial perfect empty backbone, you made sure to add lab strains of E. coli used today are descended the right tags and a nuclear localization signature from two individual isolates, the K-12 strain and (NLS) to your gene, you put a fluorescent protein the B strain. K-12 was isolated from a patient in downstream, separated by an IRES element. 1920 and eventually led to the common lab strain You did a lot of work! But let’s take a moment to MG1655, which led to DH5alpha and DH10b (also recognize the prokaryotic minions that carried known as TOP10). The history of the B strain is a out the labor-intensive process of replicating your bit more convoluted due to researchers sharing new plasmid: the Escherichia coli bacterium. and renaming samples throughout history. It was likely isolated in 1918 but was first referred to as “B strain” in 1942. The BL21 strain, (and It’s hard to count the number of different derivatives) are the most common examples of commercial strains of E. coli currently available the E. coli B strain. – a quick Google search suggests there are hundreds. This only includes general lab strains designed for subcloning or protein expression. COMMON E. COLI STRAINS USED IN If you were to include customized strains, the THE LAB number is probably in the thousands! The goal of this section is to provide enough background for Most of the commercial strains you find today you to distinguish the features of any common are marketed for a specific purpose: fast growth, lab strain and determine whether it is appropriate high-throughput cloning, routine cloning, cloning for propagating your plasmid or carrying out unstable DNA, preparing unmethylated DNA, your experiment. and more. Many mutations that make these features possible are present in most commercial strains, especially mutations that make major HISTORY OF E. COLI STRAINS improvements such as those that increase plasmid yield and/or DNA quality. Table 1 below outlines a E. coli are gram-negative, rod-shaped bacteria few of the more common genetic changes found that were named after Dr. Theodor Escherich, the in E. coli strains and Table 2.2 outlines common scientist who first described them in 1885. E. coli lab strains of E. coli. n Plasmids 101 4th Edition 39 CHAPTER 2 | BACTERIA Table 2.2 - Common Gene Mutations Found in E. coli Strains Genes Recognition Site Functional Consequence Genes Recognition Site Functional Consequence dam DNA Adenine Preparing unmethylated gal Mutation in Cells cannot grow on just methylase DNA, important when galactose galactose mutation trying to cut with certain restriction enzymes (ex: metabolism (GATC) ClaI or XbaI) pathway gyrA, DNA gyrase Confers resistance to dcm DNA Cytosine Preparing unmethylated gyrA96 nalidixic acid mutation methylase DNA, important when mutation trying to cut with certain (CCWGG) restriction enzymes that Unmethylated DNA not hsdR(rk-, mk+) are methylation sensitive. degraded, cell still can methylate DNA Increases the stability of hs - dnaJ Mutation in a dRMS chaperonin certain expressed proteins hsdS(rk-,mk-) Unmethylated DNA not gene degraded, cell cannot methylate DNA Endonuclease endA, Improves plasmid yield endA1 I (nonspecific lac Blue/white screening of Lac Operon cleavage of clones Mutations dsDNA ) mutation lacIq Lac repressor overproduced, F Host does (F’) A low copy-number expression from pLac or does not plasmid, encodes proteins repressed more (F-) contain needed for bacterial the fertility conjugation. Genes listed lacZ plasmid. on F´ are wild-type unless β-galactosidase activity indicated otherwise abolished lacY T1/T5 Phage resistance Lactose permease fhuA ferric (formerly inactivated, lactose cannot hydroxamate tonA) be taken up by cell uptake, iron uptake receptor mrcA, Inactivation Allows for uptake of mutation. mcrBC of pathway foreign (methylated) that cleaves DNA methylated cytosine DNA Plasmids 101 4th Edition 40  CHAPTER 2 | BACTERIA Table 2.2 - Common Gene Mutations Found in E. coli Strains Genes Recognition Site Functional Consequence Genes Recognition Site Functional Consequence constitutive mrr, Mutation in Cells cannot grow on deoR expression Allows uptake of large Δ(mcrC- galactose just galactose plasmids of genes for mrr) metabolism deoxyribose pathway synthesis mrr, Allows for uptake of hee N/A High electroporation Inactivation of Δ(mcrC- foreign (methylated) efficiency pathway that mrr) DNA cleaves methylated adenine or Suppression of the supE44 N/A cytosine DNA (glnV44) amber (UAG) stop codon by inserting glutamine recA, Reduces plasmid recA1, re- Mutation in a recombination, supF cA13-mrr) DNA-dependent Suppression of the increases plasmid (tyrT) N/A ATPase that is amber (UAG) stop codon essential for stability by inserting tyrosine recombination and general DNA repair λ-thi-1 Mutation Requires exogenous or thi1 in thiamine thiamine for growth metabolism recBCD Exonuclease V Increased plasmid activity abolished stability, reduced recombination Inability to utilize ara Disruption of arabinose arabinose as a carbon metabolism source relA or Relaxed Allows RNA synthesis relA1 in absence of protein pathway phenotype, mutation synthesis β-isopropyl eliminating leuB malate Requires exogenous stringent factor dehydrogenase, leucine source for inactivated growth recBCD Exonuclease V Increased plasmid mutation activity abolished stability, reduced proAB Requires exogenous in proline recombination proline source for biosynthesis pathway growth ptrc- N/A ccdA ptrc-ccdA rpsL Mutation in subunit S12 of Confers resistance to 30S ribosome streptomycin hte N/A high transformation efficiency Plasmids 101 4th Edition 41 CHAPTER 2 | BACTERIA Environmental Plasmids H By Jessica Welch | January 2017 ere at Addgene, we often refer to also harbor these circular, extrachromosomal, plasmids as lab or experimental double-stranded DNA passengers. For example, tools. They certainly are very handy a variety of high-copy number yeast vectors have in research, but where did these tools come from been derived from the natural yeast 2-micron and why do they exist in nature? In this section, plasmid. we’ll talk about environmental plasmids and how they’ve helped us develop molecular biology tools for the lab. PLASMID TRANSFER OUTSIDE THE LAB NATURAL PLASMIDS In the lab, we use chemical transformation, electroporation, and transfection to put plasmids Plasmids found in nature often give their hosts into cells. In nature, the two main routes of beneficial traits that allow them to survive in plasmid transfer are conjugation and natural competitive environments. Plasmids derived transformation. Conjugation occurs when a sex directly from the environment are sometimes pilus extends between two bacteria, allowing a called ‘natural’ plasmids, to distinguish them copy of plasmid DNA to travel from one cell to from the modified versions we usually work with another. in the lab. Horizontal (or ‘lateral’) gene transfer occurs when genetic material is passed between DNA can also be passed between bacteria by organisms by mechanisms other than reproduction bacteriophages, the viruses that infect bacteria. (vertical gene transfer). Small, mobilizable genetic Bacteriophage genomes can exist in bacterial elements like natural plasmids enable horizontal cells as extrachromosomal plasmids known as gene transfer, allowing beneficial traits encoded by these plasmids to spread rapidly throughout the environment. Indeed, plasmid transfer between organisms is one of the most common methods of horizontal gene transfer in nature. These ‘extra’ pieces of DNA can be extremely useful for survival in the crowded and competitive natural environments outside the petri dish. Although we usually think of bacteria when we think of plasmids, both Archaea and Eukaryota can Plasmids 101 4th Edition 42  CHAPTER 2 | BACTERIA prophages, or they can integrate into the host those pesky Penicillium) can be passed around chromosome using DNA recombination systems. on plasmids like a “get out of jail free card.” These Some of these systems have been modified for plasmids were historically called R (resistance) use in genome engineering, including the Cre-l