...Updated Jun 1, 2021 by Meghan Rego. Reproducible data are key to science, so scientists are used to repeating...the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from purified...serotype. Additionally, we check viral transgene expression in transduced cells whenever possible. This post... for transgene expression. Once packaged in AAV, this pre-recombined DNA could lead to transgene expression... to these expected hits, we always get hits to “random” genes. Do these hits always mean the sample is...wanted to see how low we could go. We prepared a range of spiked samples from 0.1-20% and discovered that...find the optimal dose that allows for sufficient transgene expression but limits the likelihood of recombinase-independent...

...fluorophores are constantly being engineered. Table 1 lists a few common fluorescent proteins and their ...definitions found in Table 2 may not apply. Table 1: The specific mutations comprising common fluorophores...physiological processes. Re-engineering GFP to increase its range of color and application Almost as soon as its...backbones for constructing fluorescent fusions. Transcription Reporter: Placing GFP under the control of a...promoter in a given cell type. This type of transcription reporting was among the earliest uses of GFP...GFP. Fluorescence resonance energy transfer (FRET): This is used to study the interactions between two ...ion (such as Ca2+) concentrations and pH, using a range of strategies such as FRET, calmodulin, and others...

...In Biochimica et Biophysica Acta - Biomembranes (Vol. 1838, Issue 1 PARTA, pp. 15–33). https://doi.org...principles of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes... dopamine D2/D3 receptors. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-78827-9 Breen... of fluorescence: For applications that involve ranges of pH, and significant lengths of time, the stability...receptor conformation and signaling activity​​ (Sridharan et al., 2014). It's also crucial to evaluate how...the background. Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental...considerations regarding the choice of fluorescent dyes (Sridharan et al., 2014). Manufacturers will typically recommend...

...for example, split cells on a Monday diluting them 1:10, you should be able to split on the following Thursday...Immunology. You can find Sana on LinkedIn.   References 1. Yin, R., et al. "Gene expression profiling analysis... are sensitive to pH. Anything between orange and light orange usually indicates that the cells have grown...culture media contain the pH indicator phenol red (pH range 6.0 (yellow) - 8.0 (red)). Blood pH normally lies...too young and should not be used for important transfection experiments right after they’ve been thawed.... spent organizing, an hour is earned (Benjamin Franklin)! It’s good practice to label your plates and/...

...CRISPR tools.  Distributing and depositing In Figure 1, you can see cumulative numbers of CRISPR plasmid ...new CRISPR depositors each year.    Figure 1: Addgene CRISPR distribution and deposits. (A) Cumulative...generate a classic double-stranded break, while modulators regulate transcription and chimeric efforts make...Countries shown are the top 10 requesting countries. FR=France; KR=South Korea; CH=Switzerland; AU=Australia.  ...Pyhtila et al., 2023). Our thanks to Rodolphe Barrangou for his authorial contributions to, and support...References Pyhtila, B., Kasowitz, S., Leeson, R., & Barrangou, R. (2023). The Expanding Dissemination and Distribution...

...listen to the interview with Tom McFadden!   1. Rosalind Franklin vs. Watson & Crick - Science History Rap...telling following the unfortunate story of Rosalind Franklin and her incredibly important role in solving the...

...cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR proteins (Acr's) can block CRISPR activity... ways to anti-CRISPR proteins in your experiments 1. Reduce off-target effects Prolonged Cas activity ... Knott GJ, Edraki A, Garcia B, Amrani N, Chen JS, Cofsky JC, Kranzusch PJ, Sontheimer EJ, Davidson AR,...695 . https://doi.org/10.3390/v10120695 Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki...

...editors (ABE) mediate A•T-to-G•C base changes (Figure 1), but it can be challenging to make these base changes...dCas to the genomic DNA of interest  Figure 1. When ABEs are in action they bind DNA so that the...the TadA domain can edit the short single-stranded DNA stretch that is accessible. Image from Gaudelli ... protospacer, the editing capacity of the ABE8s ranged from 1.5x higher for positions A5-A7 to 3.2x higher...References Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base ...

...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...