...folding, and deal with sub-optimal codon usage (Table 1) A few mutations are common to all or most expression...curated list of Bacterial Expression Systems Table 1: E. coli expression strains  Note: All strains are...specialized strains are available to confer greater transcriptional control, assist with proper protein folding...harboring this mutation are deficient in outer membrane protease VII, which reduces proteolysis of the...Lacks functional RNaseE which results in longer transcript half-life F- ompT lon hsdSB (rB- mB-) gal dcm...Chloramphenicol (pRARE) Good for “universal” translation; contains 7 additional tRNAs for rare codons ...pLemo (CamR) Expression of toxic, insoluble, or membrane proteins  T7 Express   IPTG-inducible expression...

...sequence on either side of the desired mutation (usually 1-3 mismatched bases) is sufficient for your primers...researcher at Cornell University.       References 1. Breslauer, Kenneth J., et al. "Predicting DNA duplex...homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires...methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the...products of the PCR reaction are pieces of double stranded, circular DNA with staggered nicks, recent data...final PCR products are pieces of linear, double stranded DNA with homologous ends. These homologous ends...the adenosine of the GATC recognition site. Transformation: After the PCR reaction, no ligation is required...

...$47,476 and the date for implementation was set for Dec 1 2016. In order to be exempted from the need to pay... these institutions (populations potentially from 1 to 5761, according to the NSF data) and therefore ...about? In March 2014, the White House issued a memorandum to the Secretary of Labor to update the FLSA,...postdocs in the final ruling was, for a lot of us, frankly quite surprising. Consortia of higher education...issued, a joint blog post by Director of the NIH Francis Collins and Secretary of Labor Thomas Perez, Fair...exactly how postdoctoral salaries are changing. Transparent salary information for postdoctoral positions... of postdocs at different points on this salary range. We have been able to gather data on changes to ...

...possible without double-strand breaks (DSBs) or donor DNA templates.  Figure 1: An overview of prime...demonstrated ~20-50% efficiency in HEK293T cells with 1-10% indels. Since then, further innovations have continued...Engineering. https://doi.org/10.1038/s41551-024-01227-1 Yan, J., Oyler-Castrillo, P., Ravisankar, P., Ward... non-edited strand, they can cause the cell to remake that strand using the edited strand as the template...the nicked target strand. The initial result is a heteroduplex, with overlapping strands of edited and unedited...Improving reverse transcriptase efficiency Building upon prior reverse transcriptase research, the Liu...the edit into one strand, there’s a mismatch between the original sequence on one strand and the edited ...

...different ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure 1: Transformation... differences between transformation, transduction, conjugation, and transfection Resources on Addgene.org...Transformation introduces transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation...bacterial transformation of chemically and electrocompetent cells is minimal (picogram range). It can ...plasmids into mammalian cells? Use transfection grade DNA. Transfection is the process of inserting an ...without commercially available kits and DNA for transformation doesn’t need to be as pure as for other uses...scientists to clone from a smaller amount of DNA (ng range). To allow efficient and flawless cloning it is ...

...forward to another great year of sharing!   References 1. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout... name suggests, lentiCRISPR v2 is a lentiviral transfer vector used to deliver both Cas9 and your gRNA...metalloproteinase-9 secretion, and identifying lysine methyltransferase substrates. We’re incredibly happy that lentiCRISPR...

...is here to help. Contact us at [email protected] or +1 617-225-9000, or check out our MTA reference pages...Uniform Biological Material Transfer Agreement (UBMTA) for its material transfers. The UBMTA was drafted by...organizations Assurance that recipient may not use the material for a commercial purpose Assurance that recipient... has time for paperwork? Here’s why a Material Transfer Agreement (MTA) matters: Science thrives on collaboration...universities and institutions require them for material transfers. When Addgene entered the plasmid sharing space...receiving institution. Therefore, many technology transfer officers are familiar with the UBMTA and are happy...  material transfer agreement...

...’ve come to the right place. Get started with PGC-1 plasmids from Addgene. References: Skeletal muscle...PGC-1a1, a transcriptional coactivator induced in skeletal muscle by exercise and endurance, protects ...have reported a mechanism based on studies of transgenic mice that helps to explain how it works. The ... skeletal muscle expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into...

... successful after their training.   Figure 1: Professional Development Hub Workshop Attendees and...ample resources and a positive perspective on the transition to a post-academic career. This training was ... inadequate job of supporting science trainee transition into diverse, successful careers.  We’re not ...components of predoctoral and postdoctoral training. A grant from the National Institute of General Medical Sciences...

... specific post-transcriptional regulation of endogenous RNA. J Biol Eng. 2014 Mar 1;8(1):7. doi: 10.1186...ability to direct enzymes – from nucleases to transcription factors – to specific sequences of DNA has become...Biological Engineering in March is fused to a translational repressor. In principle, however, designer PUF...

...improved sensors for these and other targets. Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted...fluorescent biosensors. Nat Commun. 2022 May 25;13(1):2919. doi: 10.1038/s41467-022-30685-x. Feel the ...new class of sensors for neuropeptides and neurotransmitters. Motivated to develop a biosensor for oxytocin...devised a “Modular fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA...sensors based on GPCRs recognizing various neurotransmitters, neuropeptides and other ligands - almost ...kinetics and localization for imaging synaptic transmission. bioRxiv 2022.02.13.480251. doi: 10.1101/2022.02.13.480251...2022.02.13.480251 JEDI-2P voltage sensor The François St-Pierre lab and colleagues have developed a new...

...infections, cancer, and other diseases. References 1. Harrington, Lucas B., et al. "Programmed DNA destruction...that Cas14 could indiscriminately cleave ssDNA in trans like Cas13 and Cas12 do for RNA and dsDNA, respectively...can cleave fluorescent probes of dsDNA and RNA in trans. Fluorescent signal indicates the presence of this...

...education, government, and civilization in general (1). Science and politics? Science is an apolitical process...consequences of a march. Three of these risks include: 1) Lost trust Perhaps the greatest risk is that the ...working full-time, and have received a research grant in the last five years as polled in the American...climate change page on whitehouse.gov, freezing grants, and proposing to defund aspects of research at...following subjects: Climate change Vaccines GMOs Transgender issues Reproductive health Immigration Pollution...while many also seek to honor and credit women, immigrants, and minorities who have had great impacts on...encouraged by “Broader Impacts” requirements for certain grants) (28). As one young scientist, Matthew Niederhuber...

...fluorescent in acidic organelles (Figure 1), showing that its acid tolerance is maintained in cells.   GFP is ...References 1. Zacharias, David A., et al. "Partitioning of lipid-modified monomeric GFPs into membrane microdomains...of different fluorescent protein are available (Cranfill et al., 2016 and FPbase.org). A number of variants...specific RNA stem loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can...clearly the case when proteins undergo post-translational modification with fatty acyl chains. For instance...28360214. PubMed Central PMCID: PMC5385933. 6. Cranfill, Paula J., et al. "Quantitative assessment of ...PMC2644460. 12. Querido, Emmanuelle, and Pascal Chartrand. "Using fluorescent proteins to study mRNA trafficking...

...by insertion and deletion frequency. Fig. 1: Overview of TIDE. The input is sequencing trace files...keep your product to knock-in size ratio below 10:1. Small knock-ins Is your knock-in edit small, with...optimizing this step, or consider enriching for transfected cells by flow sorting or drug selection, assuming...

...with the Gibson assembly master mix for 1 hour, and then transform into competent cells. The success rate...you should consider giving it a try! References: 1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison...generating long overhangs which allows the single stranded regions with homology to anneal a polymerase, ...hour or so, the sample is immediately ready to transform into competent cells. The master mix of enzymes...if the ends of the fragments have stable single stranded DNA secondary structure, such as a hairpin or ...would directly compete with the required single-stranded annealing and priming of neighboring assembly ...

...at a low angle (FSC) or large angle (SSC) (Figure 1). FSC values depend on the cell's size, while SSC ... or ask experienced colleagues.   Figure 1: The laser pulse illuminates the cell and is scattered...software in order to generate signals in a reasonable range. If colleagues in your group already have experience...experimentally-specific gates. Two-dimensional staining (quadrant gating) For our last example plot, I will show...cells (CD8+) from dendritic cells (CD11b+) using a quadrant gate. Within my “lymphocyte + single cell + live... two stains in relation to each other. Using a quadrant gate, I can divide my cell population into DN,...for each stain respectively). (B) Instead of a quadrant gate, I am free to use multiple individual gates...

...compare the experimental group.     Figure 1: A, Most biological experiments will benefit from ... Histochemistry & Cytochemistry, vol. 59, no. 1, SAGE Publications, Jan. 2011, pp. 6–12. Crossref,...across samples and equal protein transfer from the gel to the membrane. A loading control is critical for...identically, used to assess naturally occurring random biological variability. If one were studying the...purple) and nonspecifically through the Fc region (orange). The Preimmune serum control will not bind to .... Always probe the loading control on the same membrane that the target protein was measured on.    Flow...

...downstream experiments. You only successfully generate 1 null clone and proceed with it. In this case, the ...there are 10 putative off-target locations you can 1) determine if editing at those sites would confound...While this may effectively serve as a transfection or transduction control, a different gRNA would have..., you’ll want to select a Cas enzyme with low tolerance for mismatches within the gRNA sequence. ...near each other, this will result in a DNA double strand break (DSB), the event required for frameshift ...

...in the crystal jellyfish Aequorea victoria (Figure 1) in molecular detail. On the underside of the “umbrella...glowing protein could be used to measure gene transcription, as well as to track protein localization. And...bioluminescent, some types of corals and anemones are vibrantly colored. He hypothesized that GFP-like proteins...