...Escherichia coli bacteria. It’s hard to count the number of different commercial strains of E. coli currently...carrying out your experiment. History of E. coli strains E. coli are gram-negative, rod shaped bacteria that... the most common examples of the E. coli B strain. Common E. coli strains used in the lab Most of the... all based on E. coli K-12 and are considered BSL-1. Table 2: Lab strains of E. coli Strain Natural...+), λ– HB101 Streptomycin Hybrid of E. coli K12 and E. coli B (mostly K12, though), common lab strain... E. coli and the common features found in those bacteria. Check our our blog post covering E. coli and... changes found in E. coli strains. Table 1: Common gene mutations found in E.coli strains Gene(s) Description...

...knock-ins CasX Drug inducible CRISPR/Cas9 activation E. coli genome-wide inhibition library CRISPR knockout libraries... kit CasX, a small Cas protein, is active in E. coli and human cells Two years ago, Jennifer Doudna’s...data demonstrating that CasX is active in both E. coli and human cells. Using the structure of CasX at ...deposited plasmids for active and inactive CasX for E. coli and human expression. Read the paper in Nature ...Research Find the drug inducible Cas9 plasmids E. coli genome-wide inhibition library In the last edition...“What’s New in CRISPR,” we shared with you an E. coli genome-wide inhibition library from the Chong Zhong...Zhong lab. Now, we’ve included another E. coli genome-wide inhibition library from David Bikard’s lab....

...and Jeffrey Tabor have been working on making E. coli light responsive since 2005, when Tabor was working...lab, making light-controlled gene expression in E. coli easier and more robust. Synthetic photobiology:...light-responsive systems found in some bacteria, but not E. coli. They fall under the heading of two-component systems...light, Christopher Voigt’s lab created the first E. coli light-sensitive two-component system (TCS). Levskaya...photosensory domain from cyanobacteria to a common E. coli histidine kinase. This hybrid construct senses light...sophisticated control of gene expression. An E. coli light-sensitive two-component system (TCS). A hybrid...bulky and easier to use with other strains of E. coli, Schmidl et al. condensed the system to two streamlined...

...Escherichia coli One of the most recognizable and utilized model organisms is Escherichia coli. The bacterium...Bernardo, 2017).  Escherichia coli. Image from NIAID. E. coli is often a part of the “molecular... in E. coli have been manipulated for use in molecular biology studies. For example the E. coli biotin...Escherichia coli as a Model Organism and Its Application in Biotechnology. In Escherichia coli - Recent ...small size, and low cost, it’s understandable why E. coli is the most popular bacterial organism in microbiology...manipulation and their ability to be replicated. Because E. coli are easily propagated and have a generation time...molecular cloning and metabolic engineering.  The E. coli genome has an extensive research history and characterization...

...from a plasmid construct, most often in special E. coli strains designed for protein expression. Cultures...protein - two parameters that affect how well E. coli can produce your protein. Structural information...please see Plasmids 101: Protein Tags. Beyond E. coli: purifying from Bacillus subtilis and Pichia pastoris... for protein expression in systems other than E. coli, specifically the bacterium Bacillus subtilis and...added to proteins in other eukaryotes better than E. coli. Plasmids designed for these systems may include... bond formation. If one prefers expression in E. coli, the use of a thioredoxin A (TRX)-containing vector... Resources on the Addgene Blog Plasmids 101: E. coli strains for protein expression Plasmids 101: Protein...

...pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella ...in E. coli. However, some users pointed out that this system did not function properly in E. coli BL21(...pEcgRNA can be applied not only in coli K-12 strains, but also in E. coli B and W strains. The optimized ...pEcgRNA. Next both plasmids are transformed in E. coli with the donor DNA for editing to take place. Then... be operated at 37 ℃ for faster growth of its E. coli host, and the addition of a sacB gene renders it... arrays targeting multiple different sites in E. coli. pCutamp cures the plasmids by targeting the ampR...Yang S (2015) Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Appl Environ ...

...and E. coli codon preference. Some codons commonly used in humans are not at all common in E. coli and vice...Rosetta E. coli strains express a variety of tRNAs that are normally found at low abundance in E. coli. The ... example, during high growth rate conditions, E. coli preferentially upregulates production of tRNAs that...efficiency is well correlated with codon bias in both E. coli and S. cerevisiae. Protein folding - If a protein...genome, for instance, it may not express at all in E. coli (you can find a variety of databases showing various...dependence of transfer RNA abundance in Escherichia coli." The EMBO journal 9.13 (1990): 4359-4366. PubMed...full‐length human proteins expressed in Escherichia coli." Protein Science 19.7 (2010): 1312-1326. PubMed...

...experiments. Pick the right strain While common E. coli strains like DH5-alpha are recA-, some strains have...Recombinase-deficient strains like Stbl2, Stbl3, and NEB stable E. coli reduce the rate of recombination in samples. For...gene mutations in our Plasmids 101: Common Lab E. coli Strains blog post. Check yourself before you recA...round tan colonies that look similar to normal E. coli cultures contain the recombined vector backbone....instability-prone lentiviral vector plasmid in Escherichia coli Stbl3. 3 Biotech 3:61–70. https://doi.org/10.1007...expression vectors using reduced-genomeEscherichia coli. BioTechniques 43:466–470. https://doi.org/10.2144...strategy for efficient genome editing in Escherichia coli. Microb Cell Fact 19. https://doi.org/10.1186/s12934...

... E. coli genome as a negative control. Read the paper in Nature Communications Find the E. coli genome-wide...new CRISPR tools for your research! This time: E. coli genome-wide CRISPR inhibition Covalent tethering...Nme2Cas9 CRISPR interference in Candida albicans E. coli genome-wide inhibition library To expand the tools...tools available for genome-wide studies in E. coli, the Chong Zhang lab developed a genome-scale CRISPR...pooled library (~60,000 gRNAs) that targets ~4,000 E. coli genes with ~15 gRNAs per gene and includes ~10,000...

...strains of E. coli for DNA propagation. While great for cloning purposes, these E. coli strains are not...Systems Table 1: E. coli expression strains  Note: All strains are derived from the E. coli B strain, except...arise when over-expressing a foreign protein in E. coli. We will review the potential pitfalls of recombinant...recombinant proteins are typically expressed in E. coli engineered to accomodate high protein loads using...additional tRNAs for rare codons not normally used in E. coli. Expression vector cannot have p15A origin of replication...*, ** Kanamycin (pREP4) Cis-repression of the E. coli T5 promoter (found on vectors such as pQE or similar... e. coli strains...

...ssDNA oligos to avoid MMR: 1) E. coli with inactivated MMR: Using E. coli with inactive MMR is definitely... allows for direct modification of DNA within E. coli and is independent of restriction sites. The lambda...tags. It also has the flexibility to modify the E. coli chromosome, plasmid DNA or BAC DNA.  To use the...substrate (either dsDNA or ssDNA – see below) into E. coli expressing the lambda red enzymes. These enzymes...from digesting linear DNA introduced into the E. coli. Exo: Exo is a 5’→3’ dsDNA-dependent exonuclease...with integrated defective prophage A number of E. coli strains exist that stably express lambda red recombineering...strain is DY380, which is derived from the DH10B E. coli strain. Several other bacterial strains commonly...

... gene, located on the F sex factor plasmid of E. coli, is part of a toxin-antitoxin system encoded by ...contained the ccdB gene in frame with an MCS. E. coli that were transformed with the empty vectors expressed...investigator’s gene of interest.   CcdB resistant E. coli strains complete the system But ccdB alone isn’t...expressing it? The answer lies in special strains of E. coli that tolerate the expression of the toxin gene. ...will be present. Most common cloning strains of E. coli do not contain the F plasmid (and are considered...plasmids. Check out our blog post on Common Lab E. coli strains for more strain info. Additional cloning...on the Addgene Blog Plasmids 101: Common Lab E. coli Strains Plasmids 101: TOPO Cloning Plasmids 101:...

... making it easier than ever to use CRISPR  in E. coli and other bacterial species. Read on to learn about...the first synthetic bacterial CRISPR system in E. coli. The system available from Addgene consists of two...the gene of interest and kanamycin resistance E. coli carrying the phage recombineering machinery are ...’t the primary editing force. In contrast, in E. coli, CRISPR is primarily a means of selection that targets...protocol that requires only 2-3 hours incubation. E. coli and T. citrea scarless editing plasmids Sheng Yang...cured from the bacteria. Although developed in E. coli, the system was used successfully in Tatumella citrea... pEcCas/pEcgRNA, allows it to be used in more E. coli strains including the popular BL21(DE3) (Li et al...

...and E. coli. Like plasmids used in other systems, binary vectors are cloned and prepped in E. coli. However...as well as plasmid yield during preparation in E. coli. Addgene depositor Indu Maiti has created a new ...contain two origins of replication, one each for E. coli and Agrobacterium, along with a selectable marker...T-DNA borders to 25 bases each. By switching the E. coli origin of replication and codon-optimizing the Agrobacterium...

...suboptimal ones, which could allow even your regular E. coli to thrive in the bitter cold.  Since our objective...would be a transformed mesophilic, for example E. coli, carrying a plasmid expressing a protein of interest...consider them worth the trouble.  Enter ELSA: E. coli Learning Suboptimal Acclimatisation To resolve this...psychrophilic proteins. As the figure below shows, E. coli expressing the chaperones protein resulted in higher...higher levels of growth compared to the E. coli strain expressing the empty vector. And thus was born ...born ELSA - our happy lil’ cold-tolerant E. coli! Additionally, we’re also working towards widening the applicability...

...DNA fragments in E. coli Now that random DNA fragments have been cloned into E. coli cells, isolated colonies...fragments so that these fragments can be cloned into E. coli and subsequent proteins screened for cell-binding...vector designed for protein overexpression in E. coli, such as pET30a. If working with DNA that is refractory...resultant plasmid libraries into chemically competent E. coli and analyze transformants for appropriately sized...colonies. Transform the resultant plasmid DNA into E. coli BL21 (DE3) cells (these have the T7 polymerase and...membranes for 15 min to kill any remaining live E. coli. At this point, it can really help to incubate the...conditions. Note: if using fast-growing bacteria like E. coli or Salmonella, or any cells that bind strongly to...

...The E. coli strain Most E. coli strains can be used to propagate plasmids, but endA- E. coli are best...started: Will the plasmid be used exclusively in E. coli? Gram negative bacteria in general? Both Gram negatives...-unnatural amino acid mutagenesis in Escherichia coli. Biochemistry. 2013 Mar 12;52(10):1828-37. doi: ...

...dcm– strain of E. coli such as JM110 and re-purifying it. These specialized E. coli strains have been ...enzymes can effectively cleave DNA. Common lab E. coli K12 strains such as DH5alpha contain 3 methylases...the other hand, should be derived from a dam+ E. coli strain and will therefore have methylated adenines...Discovery of DpnI Review describing dam-/dcm- E. coli Resources on the Addgene Blog Keep up with the...

...expressing E. coli aided in ultrasound imaging of the mouse gastrointestinal tract. The E. coli strain Nissle...created two constructs optimized for expression in E. coli: acoustic gene 1 (arg1) and acoustic gene 2 (arg2...20 hours of growth following acoustic collapse E. coli re-express arg1 gas vesicles and restore their ultrasound...

...decided to add a bit of fun. He mixed together 10 E. coli strains producing different pigments, and tasked...ambient light. Expressing these chromoproteins in E. coli would make it easy to distinguish colonies from ...students transform them into different strains of E. coli,” says Westenberg. During the transformation experiment...

...cyanobacteria, synthetic inverters in E. coli, and biosensors in E. coli: Čelešnik, Helena, et al. "Biosafety...synthetic biology or protein purification studies in E. coli. 800 and 1900 Series If you’re expressing two genes... be used to precisely tune your polycistronic E. coli expression constructs. Some applications of plasmids...in the kit can be used to purify proteins from E. coli, Bacillus subtilis, or Pichia pastoris depending...aggregation propensity in four expression systems: E. coli, wheat germ, HeLa, and Leishmania (Gagoski, et al... clone your segments of choice into either an E. coli or a mammalian expression vector in a one-pot reaction...

...interaction detection (RaPID) method. RaPID borrows the E. coli biotin ligase BirA* from BioID and allows a researcher...interactions. Both methods rely on the promiscuous E. coli biotin ligase, BirA*, to biotinylate proteins that...subtilis. BASU was  compared head-to-head with the E. coli and BioID2 BirA*s in the context of RaPID for their...no detectable signal at the same time point. E. coli’s BirA* was tested at its optimum 18 hour time point... generated a >30-fold enrichment compared to E. coli’s BirA*. Faster labeling kinetics also means that...

...some E. coli strains have natural antibiotic resistances, so make sure your plasmid and E. coli strain ... makes our lives. Plasmid transformation into E. coli is a fairly inefficient process– just 1 out of 10,000...are compatible! Check out this list of common E. coli genotypes and their natural resistances.  Additional...

...highest levels of nonlinear acoustic contrast in E. coli. We deleted an unannotated hypothetical protein,... with several inducible expression systems in E. coli. The arabinose-inducible promoter pBAD and a low-copy...pBAD-bARGSer-AxeTxe, gas vesicle expression in E. coli is easy: simply grow cells in Lennox LB (10 g/L ...expression in vivo is also straightforward: after the E. coli have colonized tumors, inject L-arabinose intraperitoneally...).     Fig. 1: Expressing bARGSer in E. coli. (a) Plasmid diagram of pBAD-bARGSer-AxeTxe. (b)...imaging of tumor colonization by bARGSer-expressing E. coli. The GV-specific ultrasound signal inside the tumors...

...dsRNA-expressing E. coli that are mixed with liver paste. Prior to mixing with the liver paste, these E. coli are transformed...the  induction of T7 polymerase encoded in the E. coli genome (usually controlled by IPTG). For this IPTG-induced...expression to work, students have to use a commercial E. coli strain that is designated as DE3. For Phagocata ...eaters and refuse to eat liver paste mixed with E. coli. Thus, to get the dsRNA into Phagocata morgani and...

...Check the bacterial strain Differences between E. coli host strains can impact plasmid yield. Check the...is advisable to check out the genotype of your E. coli to ensure it is suitable for your application. We... We have previously reviewed many common lab E. coli strains on the blog, and also recommend this handy...OpenWetWare for a more extensive listing of common E. coli strains. Increase the volume Starting with a larger...to Verify Your Plasmid Learn about Common Lab E. coli Strains Additional Resources on Addgene.org Find...

...protocol) At Addgene, we use the Mix & Go! E. coli Transformation Kit and Buffer Set  from Zymo Research...available via Zymo Research. In brief, we grow our E. coli in LB to log phase, then wash and resuspend the ... protocol has worked well for us for multiple E. coli strains, including DH5alpha, XL1 Blue, TOP10, ccdB...

...response to changing pH, and they can rupture E. coli spheroplasts to release encapsulated proteins. Video...in a single operon and were first expressed in E. coli in the 1980s  (Kanabrocki et al 1986). Much later...showed that these fluorescent R bodies can rupture E. coli spheroplasts (cells which have had their cell walls...Absence of toxicity of R-body producing recombinant E. coli on paramecia. European Journal of Protistology 48...

...plasmids suitable for other model systems, including E. coli, plants, Drosophila, and zebrafish!   Why use multiplexed...Assembly or SLIC cloning methods. A BsaI-based E. coli multiplexing plasmid is available from the Koffas...supplied on a separate plasmid. Multiplexing in E. coli Koffas Lab CRISPathBrick Multiplex Plasmid: This...arrays A, B, C, AB, AC, BC, and ABC). Nielsen Lab E. coli CRMAGE Plasmids: The CRMAGE system is a fast, multiplexable...incubation. Kondo Lab multiplexed base editing in E. coli The Kondo lab expressed a cytidine deaminase fusion...LVA tag) to achieve specific point mutations in E. coli. gRNAs are delivered on a different plasmid and ...Mediated Multiplex Transcriptional Repression in E. coli.” ACS Synthetic Biology 4(9) (2015):987-1000. PubMed...

...looking at an example plasmid, let's review how E. coli normally replicate their chromosomal DNA. Chromosomal...positive and negative regulators. The genome of E. coli is about 4.6 million base pairs long and contains...re-activate DnaA. These regulatory mechanisms allow E. coli to control chromosomal replication and balance population...concentration of plasmid. Similar to replication in E. coli, regulation primarily occurs by controlling the ...controlled and has a low copy number. Like the E. coli chromosome, it has a single ori and requires the...regulate plasmid replication. For example, the plasmid ColIb-P9 encodes the protein RepZ to drive its own replication...An antisense RNA called Inc, also encoded by the ColIb-P9 plasmid, is complementary to part of the sequence...

...these broad host range plasmids all replicate in E. coli, which is the most commonly used bacteria in lab...Erwinia carotovora, E. chrysanthemi, Escherichia coli, Gluconacetobacter xylinus, Gluconobacter spp., ...herbicola, E. rubrifaciens, E. stewartii, Escherichia coli, Klebsiella spp., Legionella pneumophila, Methylophilus...Brucella spp., Caulobacter crescentus, Escherichia coli, Gluconacetobacter xylinus, Paracoccous denitrificans...promoter-based expression systems for Escherichia coli and Agrobacterium tumefaciens. 3 Biotech, 13(3),... ferrooxidans, Vibrio salmonicida, Yersinia enterocolitica, Xanthomonas campestris, X. maltophilia ...

...glycosylated proteins that cannot be produced in  E. coli or yeast cells. The only problem with baculovirus...bacterial host cell is almost always the answer. E. coli is definitely one of the most popular hosts for ...expression may be required if your protein is toxic to E. coli. Of all the vectors, pET, under the control of the...Systems Recombinant protein expression in Escherichia coli: advances and challenges Production of recombinant...

...knockout E. coli strain to assay for functional decaffeination in cells. The ΔguaB E. coli are missing... CBB5 and has been tailored for expression in E. coli. This operon is under the transcriptional control...Janthinobacterium sp. Moreover, a caffeine “addicted” E. coli strain expressing this operon can serve as a living...

...Lederberg in 1952 was noted to integrate into the E. coli chromosome in some cases. This terminology held ... plasmid, pSC102, with EcoRI and selected for E. coli transformants that were resistant to both. When ... M. Lederberg. “Sex Compatibility in Escherichia Coli.” Genetics 37.6 (1952): 720–730. PubMed PMID: 17247418...

... of eight bacterial toxin genes isolated from E. coli or Psuedomonas aeruginosa. These toxins were a mix...Gram-negative Enterobacteriaceae including in E. coli O157:H7, Shigella flexneri, Enterobacter cloacae... et al. "Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable...simplified chromosomal gene replacement in Escherichia coli." Appl. Environ. Microbiol. 81.15 (2015): 5103-5114...Recombineering) system for genome editing in Escherichia coli." Scientific reports 5 (2015): 15096. PubMed PMID...

...performed, the next step is to transform competent E. coli cells and select the positive clones. The entry ...expression clone. You will also need to use a E. coli strain sensitive to CcdB (e.g. DH5α, TOP10, Mach1...protein inhibits the growth of CcdB sensitive E. coli strains, most colonies should contain the desired...available for all kind of organisms from mammals to E. coli. Glossary of Gateway cloning vectors Vector type...

...into E. coli and plasmid assembly (re-circularization) occurs in vivo. We use XL-10 Gold E. coli, but have...plasmid in vitro, before transforming it into E. coli for propagation. However, while not widely known...successful recombination reported in all laboratory E. coli strains tested so far. The use of this pathway as...

...delight,” the very first protein they tested — E. coli expressing good ol’ EGFP — showed a slight but detectable...: Fluorescence intensity of EGFP-FlavinTag in E. coli changes in response to a ~25 mT magnet. Gray regions...magnetoresponse was clearly visible whether expressed in E. coli, in mammalian cells, or in purified form, and didn...license.   Video 1: Fluorescence movie of E. coli expressing MagLOV while a magnet is waved under ...

...remain the lovely whitish-cream color of standard E. coli. On the other hand, intact β-galactosidase produces...restriction digest. Make sure you use a proper E. coli strain (i.e. contains the lacZΔM15 mutation): XL1...the Addgene Blog Read Our Post on Common Lab E. coli Strains Learn to Use Yeast Two Hybrid Systems Browse...

...the late nineties, this was developed into an E. coli system, with the noncanonical amino acid in the ...expression of phosphoserine-including proteins in E. coli (Zhu et al., 2022). It uses the TAG codon for the...Phosphoserine into Recombinant Proteins in Escherichia coli. 12(21). https://doi.org/10.21769/BioProtoc.4541...

...elements required for plasmid maintenence within an E. coli cell, but vectors can be widely utilized across ...necessary components to allow shuttling between E. coli and yeast cells. To be useful in the lab, the vectors...Can this be used for auxotrophic selection in E. coli? Additional considerations HIS3 L-hisitidine no...

... other organisms. If you’ve ever wondered why E. coli is safe to use in the lab but also causes outbreaks..., Gateway cloning relies on genes found on an E. coli fertility plasmid that also encodes a toxin, CcdB...to that toxin, CcdA. Knowing which strains of E. coli contain this F plasmid is critical for successful...Some bacteria possess plasmid-borne toxins like colicins and subtilisins to kill other bacteria in the ...plasmids carrying genes for bacterial toxins like colicin typically also carry the necessary resistance genes...survive their own secretions. Pro tip! Historically, colicin-producing plasmids have been referred to as “Col...

...transform the PCR products into E. coli. Ligases within the E. coli then fuse together the PCR products...inexpensive DpnI enzyme. Otherwise you let your E. coli do the work for you. Traditional restriction enzyme...

... plasmid DNA extraction and purification from E. coli is the presence of the protein endonuclease I encoded...encoded by the gene endA. Nowadays, the most common E. coli strains used for plasmid amplification are endA ...

...library is transformed into E. coli for amplification. Transformed E. coli are allowed to grow on plates...is similar to amplifying a plasmid: transform E. coli with the pooled library DNA, allow the bacteria ...

...different strains of E. coli that can be used for transformation. The preferred E. coli strains for storage... has been received and stored, it is put into E. coli strains that have been treated to easily take in...

...transformed directly into chemically competent E. coli and transformants selected with the appropriate ...have been isolated from methylation competent E. coli for this procedure to work properly. Directly transform...

...chromatography. Purification of His fusion proteins from E. coli is generally more straightforward and of a higher...a Solubility Enhancer. In P. E. Vaillancourt, E. coli Gene Expression Protocols (Vol. 205, pp. 99–118)...more!   Resources and References   References Coligan, J. E., Dunn, B. M., Speicher, D. W., & Wingfield...

.... Max Juchheim in this post. 3. Plasmids 101: E. coli Strains for Protein Production Addgenie Julian Taylor-Parker...explains why you might want to use a specialized E. coli strain for protein production and gives a quick ...

...acids E. coli and Mammalian Cells pEVOL-pAzF synthetase for p-azido-l-phenylalanine E. coli pEVOL-...proven most effective only in a few cell types (E. coli and Xenopus oocytes, mainly). Nevertheless, tRNA...pEVOL-pBpF synthetase for p-benzoyl-l-phenylalanine E. coli pAcBac1.tR4-MbPyl pyrrolysyl-tRNA synthetase Mammalian...