...pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene for counterselection to yield...complete editing process. The MUCICAT system While the above systems are great for experiments that require only...serve to interrupt genes (or gene clusters) in metabolic engineering. So far, we’ve used the MUCICAT system...when compared to the industrial strain. In our metabolic engineering case study of N-acetylglucosamine ...Multiple induction systems to choose from. Ready for metabolic engineering with improved cargo dedicated to gene...other CRISPR methods for bacteria Find all articles about microbiology Resources on Addgene.org: Visit the...

... restraining further replication.   Figure 3: Comparison of replication regulation mechanisms of... negative regulators. The genome of E. coli is about 4.6 million base pairs long and contains a single...copy number and compensating for the additional metabolic cost imposed on the host with a beneficial function.... On the other hand, they also impose a lower metabolic cost on cells and produce lower amounts of protein...

...biotechnology 16(7): 657–62. PubMed PMID: 9661200.  3. Cardin, Jessica A. et al. 2009. “Driving Fast-Spiking... on the Addgene Blog Learn More about FLEX-vectors Learn More about Cre-Lox Check out Retrograde AAVs...expression have been developed. Read on to learn more about FLP-FRT, Cre-lox, and how combinations of FLP and...

...visualized and sorted via flow cytometry. Figure 3: Targeting pEF-BFP with a cytidine deaminase base ...for base-editing activity The reporters mentioned above all rely on GFP fluorescence and cell sorting using... Additional resources on the Addgene blog Read about other CRISPR reporters Browse our CRISPR blog posts...

...types can be found on Addgene’s website. Fig. 3: Cas9 repressors include dCas9 alone and a dCas9-KRAB... 101 Series Learn about the PAM Requirements of Different Cas9 variants Learn about CRISPR Pooled Library...

...Biotechniques 35.4 (2003). 812-4. Pubmed PMID: 14579747. 3. Drexler, H.G., Uphoff, C.C. “Mycoplasma contamination...Lentiviruses are a powerful laboratory tool often employed to establish cell lines that stably express...Get Tips for Titering Your Lentivirus Learn All About Viral Vector Elements Learn How to Do Genome-Wide...

..." Gene therapy 6.7 (1999). PubMed PMID: 10455443. 3. Hindson, Benjamin J., et al. "High-throughput droplet... Addgene.org Find All Available AAV Learn More about Virus Production Find Viral Vector Protocols ...

...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr...different buffers and do not use water). Microwave for 1-3 min until the agarose is completely dissolved (but...cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins. Optional... of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample... of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration ...

...Model for Regeneration and Aging. Gerontology, 66(3), 212–222. https://doi.org/10.1159/000504294 Wang,... more about model organisms in the first Five Popular Model Organisms blog post Read more about plasmids...characteristics that make them ideal for studying in a laboratory environment. We’ve covered five model organisms...have been specifically developed to thrive in laboratory settings. One of its most attractive features...minutes, they are perfect for molecular cloning and metabolic engineering.  The E. coli genome has an extensive...address many areas of microbial research like metabolism. Transport, biofilms, and much more. Because ...characterized genome, and is easily maintained in a laboratory environment. What sets Arabidopsis apart from...

...thomas-kilmann-conflict-resolution-model/, accessed Jan. 3, 2022.     Additional Resources on the Addgene blog...rely on what I know about those particular teams at that given time. I think about how previous iterations...disagreements. In fact, conflict resolution isn’t just about resolving conflicts between other people, but resolving...normally get along well with each other disagree about a specific decision, direction, or goal. Resolving...of lapsed communication. Making some assumptions about how something should be communicated is necessary...confusion around... Ownership: Misunderstandings about who is responsible for what tasks or projects is...conflicts.  Transitioning a conflict into a collaboration may not be the easiest resolution to accomplish...

...strain. F- Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 λ- e14- mcrA0 relA1 rpsL150(strR) spoT1...thiamine metabolism Requires exogenous thiamine for growth ara disruption of arabinose metabolism pathway...little prokaryotic minions that carried out the labor-intensive process of replicating your new plasmid... Phage resistance gal Mutation in galactose metabolism pathway Cells cannot grow on galactose only ...repressed more   LacZ β-galactosidase activity abolished   lacY Lactose permease inactivated, lactose...plasmid stability recBCD Exonuclease V activity abolished Increased plasmid stability, reduced recombination...

...recognizes a different PAM site and generates 5’ and 3’ staggered ends after dsDNA breaks. The Doudna lab...Addgene Blog Learn about using toehold switches to detect Zika virus   Learn more about targeting RNA with...time-consuming, and complex to use outside of standard laboratories. In the case of the COVID-19 pandemic, qPCR ...with Cas13a (C2c2) Learn more about Cas12a (Cpf1) and its multiplex genome editing abilities Resources...

...Sciences, 24.9 (1999b): 359–363. PubMed PMID: 10470036. 3. Said, H. M. “Biotin: The forgotten vitamin.” American...uses of biotinylation in molecular biology! Learn about in vivo biotinylation of bacterial fusion proteins...covalently attached to the active site of key metabolic enzymes including biotin carboxylase and decarboxylases... organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier from one compound...such as gluconeogenesis, lipogenesis, amino acid metabolism and energy transduction. Biotinylation is the...

...protein." (1998): 509-544. PubMed PMID: 9759496.  3. Buckley, Anthony M., et al. "LOV-based reporters ...deleting residues. Only about 10 amino acids can be deleted from the C-terminus and about 5 from the N-terminus...cylinder with a length of 4.2 nm and a diameter of about 2.4 nm (Hink et al., 2000). The complete beta-barrel...based on cofactor binding proteins as described above. The smallest tags are peptides that are engineered...

...base editor development going forward. Figure 3: Mechanism of gGBE. Created with BioRender.com. ...In our last post, we talked about the first base transversion editors: CGBEs, or C → G Base Editors. ...apyrimidinic sites and abbreviated as AP sites. A collaborative team from the David Liu and Dali Li labs reported... CRISPR 101: Cytosine Transversion Editors Read about four base editor reporters Resources on Addgene.org...

...BioTechniques 2015; 58:161-170. PubMed PMID: 25861928. 3. Jiang W, Zhao X, Gabrieli T, Lou C, Ebenstein Y, ...the annealed and filled-in gaps The great part about this mix of enzymes is that they can all work at...the standard Gibson assembly techniques described above, this resulted in the direct and seamless cloning...Blog Browse All of Our Plasmids 101 Posts Read About Other Cloning Techniques Additional Resources on...

...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies, it...colonies and check them for successful ligations. Pick 3-10 colonies depending on the number of background ...Information about plasmid cloning by restriction enzyme digest (subcloning), including design and experimental...window) New England Biolabs for more information about restriction enzyme buffers). If you select enzymes...

...VWR, EX0276-1 Benzonase 250 U/µl, Millipore #71205-3 Polybrene 10 mg/mL, Millipore, TR-1003-G Molecular...mL glutaGRO 50 U/mL benzonase: 15 mL DMEM Complete 3 µL of 250 U/µL benzonase Procedure Transducing Cells...0.03887375114 6.22E+06 2 400 768 18360 0.08366013072 6.69E+06 3 200 1620 15840 0.2045454545 8.18E+06 4 100 3180 20540...the titer. Calculations In the experimental setup above, the following virus dilutions were used to transduce...v = Virus volume, mL *In the experimental setup above, the cells per well is 300,000 and the virus volume...

...predict which one will work as illustrated in figure 3 (Mastop et al., 2018). Unbiased, random insertions...limitations is the size of GFP: ~240 amino acids or about 28 kDa. GFP and its homologues have a beta-barrel...

...spend the next 6 years. Know yourself I rotated in 3 labs before choosing a graduate school lab. All the...matters Yes, yes it’s all about the science but really it’s not. It’s about the atmosphere that will allow...and are nervous about discussing data. There’s hesitation before answering questions about lab culture. ... emotions and assumptions. I’m not just talking about simple things like “Which route should I take to... and not very communal. That didn’t suit my collaborative learning style at all. Finally I rotated in ... did). Some of the information you will need is about you. It’s worth taking time to honestly evaluate...lab members can give open, revealing information about the culture and lab head. While it’s good to talk...