...transformed E. coli . Over-agar spreading of antibiotic makes it easy for an investigator to conveniently...stock solution in sterile water (or other antibiotic) E. coli transformed with a plasmid containing the carbenicillin...fully absorb. During the incubation, transform DH5α E. coli cells by heatshock with the plasmid of interest...transformation protocol. Plate 50 µL of transformed E. coli /rescue media suspension onto the agar and gently...carbenicillin, plated over-agar for selection of transformed E. coli . Sample Data Selection of E.coli on LB-agar...Plate with No Carbenicillin Plate shows a lawn of E. coli and no selection. 150 µL of 0.1 mg/mL Carbenicillin...Carbenicillin plated over-agar Plate shows a lawn of E. coli and no apparent selection. 150 µL of 1 mg/mL ...

...Screening for inserts E. Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol for producing...secondary structure of the hairpin sequence. Back to Top E. Producing Lentiviral Particles Before this step, ...cells that stably express your shRNA of interest. E.1 Recommended Materials Material Vendor and catalog...information, visit Addgene’s Mammalian RNAi Tools page. E.2 Protocol for Producing Lentiviral Particles This...tube. Incubate for 5 minutes at room temperature. e. Add 80 μL of FuGENE® master mix to each tube from...appropriate puromycin-containing media to cells. Days 3+: e. Examine cells each day and change to fresh puromycin-containing...polybrene. c. Add lentiviral particle solution from step E. For a 6 cm target plate, add between 0.05-1 mL virus...

...Lisowski, L., Alexander, I. E., & Logan, G. J. (2019). A User’s Guide to the Inverted Terminal Repeats of Adeno-Associated...doi.org/10.1089/hgtb.2016.133. PMID: 28967288. Shitik, E. M., Shalik, I. K., & Yudkin, D. V. (2023). AAV- based...elements, but among the most important are the inverted terminal repeats (ITRs). ITRs are the elements...2020). Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene...Merten, O.-W., & Galibert, L. (2017). Impact of Inverted Terminal Repeat Integrity on rAAV8 Production ...2017). Deletion of the B-B’ and C-C’ regions of inverted terminal repeats reduces rAAV productivity but...

...4 empty NTC C AAV reference Sample 3 D E Sample 1 Sample 4 F E Sample 2 Sample 5 F Perform data analysis...Standard curve: R 2 (coefficient of correlation) ~ 1.0, E (efficiency of PCR) ~100% (90%–110% range is acceptable...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods...

...found in E. coli have been manipulated for use in molecular biology studies. For example the E. coli biotin...small size, and low cost, it’s understandable why E. coli is the most popular bacterial organism in microbiology...2017).  Escherichia coli. Image from NIAID. E. coli is often a part of the “molecular biologist ...manipulation and their ability to be replicated. Because E. coli are easily propagated and have a generation ...molecular cloning and metabolic engineering.  The E. coli genome has an extensive research history and...2012) and APEX (Rhee et al., 2013). Research using E. coli has the potential to address many areas of microbial...biofilms, and much more. Because of it’s BSL-1 status, E. coli can be used as a model to relate these processes...

... out” of ccdB with the investigator’s gene of interest.   CcdB resistant E. coli strains complete...ccdB gene, located on the F sex factor plasmid of E. coli, is part of a toxin-antitoxin system encoded...pKIL19, contained the ccdB gene in frame with an MCS. E. coli that were transformed with the empty vectors...expressing it? The answer lies in special strains of E. coli that tolerate the expression of the toxin gene...ccdA will be present. Most common cloning strains of E. coli do not contain the F plasmid (and are considered...recombinant plasmids. Check out our blog post on Common Lab E. coli strains for more strain info. Additional cloning...Resources on the Addgene Blog Plasmids 101: Common Lab E. coli Strains Plasmids 101: TOPO Cloning Plasmids ...

...-Cortés, A. E., Walker, M., Kalmbach, B., Lenz, G. H., Morin, E., Nguyen, T. N., Garren, E., Bendrick,...Hess, E. E., Mahoney, J. T., Kojima, Y., Ding, Y., Somasundaram, S., Miller, J. A., Kalmbach, B. E., Radaelli...Taskin, N., Goertsen, D., Shay, T. F., Sullivan, E. E., Huang, S.-F., Ravindra Kumar, S., Arokiaraj, C...10.1038/s41592-020-0799-7 Shay, T. F., Sullivan, E. E., Ding, X., Chen, X., Ravindra Kumar, S., Goertsen...Ibrahim, L. A., Mastro, K. J., Sabri, E., Huang, S., Favuzzi, E., … Dimidschstein, J. (2020). Viral manipulation...., Deverman, B. E., & Gradinaru, V. (2017). Engineered AAVs for efficient noninvasive gene delivery to...//doi.org/10.1038/s41565-023-01419-x Deverman, B. E., Pravdo, P. L., Simpson, B. P., Kumar, S. R., Chan...

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

... pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella...systems in E. coli. However, some users pointed out that this system did not function properly in E. coli ...in pEcgRNA. Next both plasmids are transformed in E. coli with the donor DNA for editing to take place... can be operated at 37 ℃ for faster growth of its E. coli host, and the addition of a sacB gene renders...applied not only in coli K-12 strains, but also in E. coli B and W strains. The optimized plasmid curing...crRNA arrays targeting multiple different sites in E. coli. pCutamp cures the plasmids by targeting the... Image from Li et al., 2021.   The updates involved the following changes. In pCas9, we 1) replaced...

...Agrobacterium and E. coli. Like plasmids used in other systems, binary vectors are cloned and prepped in E. coli....infection, as well as plasmid yield during preparation in E. coli. Addgene depositor Indu Maiti has created a ... 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... (>10 kb.) Since transformation efficiency is inversely proportional to size, and many plant scientists...

...method in E. coli, Jesse Zalatan’s lab used an approach to identify known proteins involved in the activation... are making it easier than ever to use CRISPR  in E. coli and other bacterial species. Read on to learn...design the first synthetic bacterial CRISPR system in E. coli. The system available from Addgene consists ...targeting the gene of interest and kanamycin resistance E. coli carrying the phage recombineering machinery ... isn’t the primary editing force. In contrast, in E. coli, CRISPR is primarily a means of selection that...protocol that requires only 2-3 hours incubation. E. coli and T. citrea scarless editing plasmids Sheng...subsequently cured from the bacteria. Although developed in E. coli, the system was used successfully in Tatumella...

...switch in cyanobacteria, synthetic inverters in E. coli, and biosensors in E. coli: Čelešnik, Helena, et al...synthetic biology or protein purification studies in E. coli. 800 and 1900 Series If you’re expressing two... can be used to precisely tune your polycistronic E. coli expression constructs. Some applications of ...2388-2393. Huang, Shijie, Xuechen Zhu, and Charles E. Melançon III. "Detection and Quantification of Ribosome...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, ...quickly clone your segments of choice into either an E. coli or a mammalian expression vector in a one-pot...

...suboptimal ones, which could allow even your regular E. coli to thrive in the bitter cold.  Since our ...example would be a transformed mesophilic, for example E. coli, carrying a plasmid expressing a protein of ...ever consider them worth the trouble.  Enter ELSA: E. coli Learning Suboptimal Acclimatisation To resolve...psychrophilic proteins. As the figure below shows, E. coli expressing the chaperones protein resulted in... in 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...collaborative environment for research.  We’ve also involved ourselves in extensive outreach programs to encourage...

...Ferenczi, E. A., Ramakrishnan, C., O’Shea, D. J., Prakash, R., Gunaydin, L. A., Hyun, M., Fenno, L. E., Gradinaru.../10.1007/978-1-0716-0755-8_2 Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., & Deisseroth, K. (2005)..., J. C., McKnight, D. J., Yoshizawa, S., Kato, H. E., Ganguli, S., & Deisseroth, K. (2019). Cortical layer–specific...Berthold, P., Ollig, D., Hegemann, P., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated...doi.org/10.1073/pnas.1936192100 Tichy, A.-M., Gerrard, E. J., Sexton, P. M., & Janovjak, H. (2019). Light-activated...Ahlbeck, J., Augustin, S., Sauter, K., Papagiakoumou, E., Gottschalk, A., Soba, P., Emiliani, V., Engel, A...

... expressing E. coli aided in ultrasound imaging of the mouse gastrointestinal tract. The E. coli strain...created two constructs optimized for expression in E. coli: acoustic gene 1 (arg1) and acoustic gene 2 ...with 20 hours of growth following acoustic collapse E. coli re-express arg1 gas vesicles and restore their...arg1 vesicles. Following collapse, vesicles are invisible to ultrasound, which allows for differentiation...Shapiro, M.G. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts....

...failed experiments. Pick the right strain While common E. coli strains like DH5-alpha are recA-, some strains...Recombinase-deficient strains like Stbl2, Stbl3, and NEB stable E. coli reduce the rate of recombination in samples....common gene mutations in our Plasmids 101: Common Lab E. coli Strains blog post. Check yourself before you...the round tan colonies that look similar to normal E. coli cultures contain the recombined vector backbone...viral vector protocols Learn about the common lab E. coli strains Find tips for viral transduction  ...most frequent problems that I help troubleshoot involves viral vectors and plasmids with repeating sequences...

...dsRNA-expressing E. coli that are mixed with liver paste. Prior to mixing with the liver paste, these E. coli are...through the  induction of T7 polymerase encoded in the E. coli genome (usually controlled by IPTG). For this...expression to work, students have to use a commercial E. coli strain that is designated as DE3. For Phagocata...picky eaters and refuse to eat liver paste mixed with E. coli. Thus, to get the dsRNA into Phagocata morgani...are 11 different genes cloned into plasmids to investigate for each of these planarian species. These plasmids...time or after genetic manipulation. The technique involves using synthetically made RNA (riboprobes) with... the riboprobes are created, the WISH protocol involves preparation of the tissue sample, several washes...

...dam–/dcm– strain of E. coli such as JM110 and re-purifying it. These specialized E. coli strains have ...restriction enzymes can effectively cleave DNA. Common lab E. coli K12 strains such as DH5alpha contain 3 methylases... on the other hand, should be derived from a dam+ E. coli strain and will therefore have methylated adenines...Methylases Discovery of DpnI Review describing dam-/dcm- E. coli Resources on the Addgene Blog Keep up with...protomoting or repressing protein expression. Methylases involved with these processes (for example Dam and Dcm ...

... highest levels of nonlinear acoustic contrast in E. coli. We deleted an unannotated hypothetical protein...bARGSer with several inducible expression systems in E. coli. The arabinose-inducible promoter pBAD and a...With pBAD-bARGSer-AxeTxe, gas vesicle expression in E. coli is easy: simply grow cells in Lennox LB (10 ...expression in vivo is also straightforward: after the E. coli have colonized tumors, inject L-arabinose intraperitoneally.... 1c).     Fig. 1: Expressing bARGSer in E. coli. (a) Plasmid diagram of pBAD-bARGSer-AxeTxe....imaging of tumor colonization by bARGSer-expressing E. coli. The GV-specific ultrasound signal inside the...Bourdeau, R. W. et al. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts....

...incubate Check the bacterial strain Differences between E. coli host strains can impact plasmid yield. Check...it is advisable to check out the genotype of your E. coli to ensure it is suitable for your application...application. We have previously reviewed many common lab E. coli strains on the blog, and also recommend this ...OpenWetWare for a more extensive listing of common E. coli strains. Increase the volume Starting with a...How to Verify Your Plasmid Learn about Common Lab E. coli Strains Additional Resources on Addgene.org...with repeat elements, such as viral plasmids with inverted terminal repeats. We use NEB Stable to transform...