...10.1073/pnas.0909910107 Varani G, McClain WH (2000) The G·U wobble base pair. EMBO Rep 1:18–23 . https://doi.org... cerevisiae (Tuller et. al., 2010). Figure 1: An overview of peptide synthesis. A ribosome interacts...is codon usage important? Translation and protein synthesis relies on transfer RNAs (tRNAs). tRNAs bind...rate of translation and thus protein expression. In fact studies have shown that translational efficiency...on an organisms codon usage bias to increase translational efficiency and thus protein expression without...mammalian cells. This was the first codon optimized membrane protein expressed in mammalian cells. In this ...conformation, folding and stability, change post-translational modification sites and even affect protein function...

...advancing the field. References: 1. Internal initiation of translation of eukaryotic mRNA directed by a... of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. Kim et al...discovered in positive single-stranded RNA viruses to allow for the efficient translation of multiple genes from...Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single translation event ...scientists use a multitude of techniques including co-transfection of two or more plasmids, the use of multiple...Unlike promoters which will create unique mRNA transcripts for each gene that is expressed, multicistronic...your gene as they are both derived from the same transcript. Of course multicistronic vectors do not have...

... depending on the specific edit.   Figure 1: Choosing the best PE6 variant for your application...modular envelope design. Nature Communications, 14(1), 5141. https://doi.org/10.1038/s41467-023-40788-8...also incorporated LOV-Turbo into TransitID experiments.  Find TransitID plasmids here! These two developments...genetic cargo Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker ...in vitro and in vivo. Incorporating reverse transcriptase (RT) variants, PE6a and PE6b are 500–800 bp ...be tethered on the viral surface, expanding the range of targetable cell types. DIRECTED is compatible...37612276.   Optogenetic proximity labeling and TransitID By Mike Lacy Proximity labeling can identify protein...

...systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir (small ... tag size, which can also be handy!   Fig 1. dTag molecules induce dimerization of an FKBP12F36V...of an exogenous small molecule). Degron tags can range in size from several kDas up to ~35 kDa and are ... because the system modulates protein post-translationally, unlike many other methods. Secondly, these...

...sequence derived either from p63(1-29) and Lck. Image from Chertkova et al., 2020. In their BioRxiv post...Reports. 2020. https://doi.org/10.1038/s41598-020-57500-1 The CRISPR corner By Jennifer Tsang Here are a few...protease allows a transcription factor to enter the nucleus and modulate transcription of the mCitrine ...HeLa cells were transfected with plasmids encoding FPs tagged with the plasma membrane targetin sequence...non-specific labeling. For labeling the plasma membrane, they tested different target sequences for improved...expression can vary significantly depending on transgene design and delivery. To overcome these variations...into genomic DNA with the help of the piggyBa transposase. Image from Hazelbaker et al., 2020. The lab...

...be selected for these small ligands. References 1. Ma, Hanhui, et al. “Multicolor CRISPR labeling of...Biological Applications.” Sensors (Basel). 2012; 12(1): 612–631. PubMed PMID: 22368488. PubMed Central PMCID...fluor. Transcribing and translating a protein fluorophore also takes time - bypassing translation altogether... could be screened for their affinity to a wide range of non-nucleotide targets. These RNA molecules, ...The key to aptamers’ flexibility is their broad range of targets, including proteins, peptides, amino ...ions, and cells (even pathogenic bacteria). That range is possible because aptamers can either fold to ...biosensors. Aptabeacons Fluorescence resonance energy transfer (FRET) technology has been studied and used in...

...Time for the #BioSci3319 #StreakPlateChallenge part 1. Lab B needs a redo :-( @SandTBioSci #CompetitiveStreaking...and spread over the second quadrant. The process continues until all quadrants are used, hopefully revealing...students transform them into different strains of E. coli,” says Westenberg. During the transformation experiment...plate, first the bacteria are inoculated over a quadrant of an agar plate. Then, a stick is dragged through...revealing single, isolated colonies in the last quadrant after incubation. The challenge is usually successful...genetic backgrounds of the host strains affect transformation efficiency.  Aside from the Streak Plate Challenge...

...existing protein structures, called Top7. Figure 1: Top7 receives the Nobel Prize. Created with BioRender.com...References Kuhlman, B., Dantas, G., Ireton, G. C., Varani, G., Stoddard, B. L., & Baker, D. (2003). Design...

... to the development of resistance include (Figure 1): Over-prescription of antibiotics (for example, ... communication, and public health.     References 1. O'Neill, J. "Tackling drug-resistant infections globally...methods of ‘horizontal gene transfer’. Resistance can also arise from random mutations in the genetic sequence...didn’t develop, either through random mutation or horizontal gene transfer, means to defend themselves from...bacterium sharing genes with another, an accidental transmission of genes from one bacterium to another through...

...true reproducibility. The following are critical: (1) connectivity to other data sources used in research...engagement with the Wisconsin research community were to: (1) prevent vendor lock in by making it easy to get data...that emerged from intensive engagement with a wide range of people at the University of Wisconsin in 2011...reproducibility an ELN should have integrations with a broad range of relevant generic and science-specific tools. ... however, in some cases it would be possible to grant those wanting to understand and/or attempt to reproduce...Massachusetts, and feels privileged to be part of the vibrant life sciences research and commercialization community...

...more applications.   Fig. 1 Making a Protein Tag (Abridged) (1) Locate a promising monoclonal antibody...an amide group added to its C-terminus post-translationally, which turned out to be critical for antibody...

...does not greatly alter the size of the tag.   Table 1: Common protein tags Tag Epitope Mass (kDa) Function... conditions FLAG DYKDDDD or DYKDDDDK or DYKDDDK 1 Affinity and Purification Good for antibody-based ...usually smallish peptides incorporated into a translated protein. As depicted in the accompanying cartoon...peptide-based proteosome inhibitors, glutathione S-transferase (GST), which can be fused to recombinant proteins...

...dark affinity range (130nM to 4.7µM for iLID nano and 800nM to 47µM for iLID micro; Figure 1). With this... August 20th; 14(1). TRICK: A method for visualizing the first round of translation in living cells ...light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2). In the dark, this peptide is caged by the...Figure 2). They are all available now at Addgene. 1 Lungu et al., Chem Biol. 2012 Apr 20;19(4):507-17....Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; 112(1):112-7. doi: 10.1073/pnas. CRISPR-Cas9 optogenetic...for a variety of experimental purposes including (1) single or multiple, constitutive or inducible, gene...the years. The authors optimized these systems by: 1) decreasing the number of plasmids required for each...

...fragments of these transcripts. I remember when small RNA was just something we ran off the bottom of ...viruses - 2 cas systems regulate at the DNA level and 1 at the RNA level. Udi Qimron from Tel Aviv University...’t give tRNAs much thought. High throughput transcriptome sequencing methods including work done by the...hypothesized that life started with RNA and a simple cell membrane. His recent work shows that they are making fairly...knew…I had barely heard of these). These single stranded, covalently closed RNAs seem to be conserved in...

...single neurons in the living brain   Figure 1: Plasmid mix to label neuronal morphology (eYFP) and...genOway press release for more information. References 1. Atasoy, D., Y. Aponte, H. H. Su, and S. M. Sternson...concentration of FLEx vectors guarantees that most cells are transfected with all labeling plasmids and...PMCID: PMC2593125. 2. Buchholz, F, P O Angrand, and A. Francis Stewart. 1998. “Improved Properties of ... sites can lead to excision, inversion, and translocation of DNA in a similar fashion as between loxP ... has a temperature optimum of 37 °C (Buchholz, Angrand, and Stewart 1998). FLPe’s performance was further...in FLPO, which shows similar efficiency to Cre (Kranz et al. 2010). These differences in recombinase efficiency...

...Addgene's founders Podcast breakdown Intro Chapter 1 - Who are Addgene's Founders? Chapter 2 - The process...Chapter 3 - Addgene as a leader in standardized tech transfer and the adoption of the UBMTA Chapter 4 - Developing...ensuring that success Chapter 6 - Developing Addgene’s Brand and culture Chapter 7 - Building Addgene as a global...

...Figure 1). Clustered events are called a population.                             Figure 1: While...conceptualize where the populations are, try drawing a quadrant over the plot that divides it into four, using...different populations by imagining (or drawing) a quadrant over the dot plot. Created with biorender.com....Biorender! While a helpful visualization tool, a quadrant will often be too clean for real-world data. Most...and debris. The side scatter roughly measures granularity, a proxy for nucleic complexity of the cell.  ...these populations, I suggest using the imaginary quadrant to help (Figure 5). Even though this real-world...real-world data is far messier than biorender plots, quadrants can still help me as a reader if I'm struggling...

...deficient in DNA cutting activity to a transcriptional activation domain (Fig 1). Two ways to use CRISPRa for... (2016) Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems. PLoS...promoter of another gene, thereby driving aberrant transcription. That being said, we generally pick guides...been significant work on constructing Cas9 transcriptional activators. These constructs allow for the ...picking whichever guides are closest to the transcription start site (TSS). We recommend targeting the...Church GM (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12:326–328 . https...Nishimasu H, Nureki O, Zhang F (2014) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex...

...Spaghetti Monster probes at Addgene Uses of smFPs 1. Multi-channel connectomic tracers Fluorescent proteins... expanded use of smFPs in the future. References 1. Viswanathan, Sarada, et al. "High-performance probes...ideal probe should combine the solubility, cell tolerance, and optional fluorescence of FPs with facile ...

...know that most cephalopods die after they lay their 1 clutch of eggs? Propagation is thus quite difficult...organism blog posts: Five Popular Model Organisms, Part 1 Five Popular Model Organisms, Part 2 ...species. Discovering ways to culture, propagate, transform, store, and share all of these new organisms continues...Phenotype thus making progress on the NSF “G to P grand challenge.” Creating a productive, new scientific...evolution because electric organs arose in different branches of the evolutionary tree at least 6-7 times and...mushrooms) but there are over 1,000 species. No transformation techniques are available yet. Polistes (Amy... a system using novel vesicles to efficiently transform these cells which don’t like foreign DNA at all...