... (green) integrates downstream of the glmS gene in the recipient's genome. Antibiotic plates lacking DAP...bacteria. Once transferred, Mobile-CRISPRi stably integrates into the genome and is propagated during normal... a tri-parental mating scheme. Mobile-CRISPRi integrates downstream of the highly conserved glutamine-...is transferred to recipient bacteria where it integrates into the trnS-leu2 locus.   Figure 2: ...recipient bacteria by bi-parental mating. The ICE integrates into the trnS-leu2 locus. Antibiotics select ...of transfer was variable. They measured transfer rates by comparing the number of colonies that grew on...that grew on a non-selective plate. Most bacteria tested had 1 transfer in every ~100 to 1,000 colonies,...

...Protease recognition sites commonly used with tags Protease Recognition site Notes TEV ENLYFQS Cleaves...make something the cell doesn’t need to make. Eukaryotes and some bacteria deploy proteosomes to degrade...proteolysis. That’s one form of instability. Prokaryotes can also have a hard time folding eukaryotic ...correctly, there’s no point in crystallizing it or testing its function. Small ubiquitin-related modifier ...protein tags Tag Epitope Mass (kDa) Function Notes CBP KRRWKKNFIAVSAANRFKKISSSGAL 4 Affinity and ...solulibility and folding of eukaryotic proteins in prokaryotes; single step purification with amylose, but wicked...amino acid protein  12 Stability At N-terminus, promotes folding and structural integrity; cleavable. Not...

...96-well microplates, and thanks to our dedicated product and development teams, frequent updates allowed...have evolved over the years to keep up with the latest tools. In Addgene’s early days, we would ‘spot-...eventually outpaced our software, even with continuous updates. The generous donation of a Illumina MiSeq sequencer...and optimize our pipeline. After reviewing and testing several platforms for engineering pipelines, we...other programs running on a computer, so it safely operates in isolation, and can be easily shared with others...others without any complicated setup. We created, tested, and debugged each of our pipeline steps within...resources. Now, we could iterate our development and testing cycle faster due to the ease of jumping back and...

...samples of AAV for testing purposes. To address this, we recently released our serotype testing AAV, which are...GCaMP6s. jGCaMP7f is the fastest responding variant. jGCaMP7b exhibits the brightest resting fluorescence ...AAVs include: EGFP-expressing AAV for serotype testing Calcium sensors and GABA sensors Optogenetics Recombinases...are distributed individually. See our Serotype Testing AAV inventory for the entire collection! We are... promoters and fluorophores for validating and testing purposes. We will continue to add more controls...calcium sensors and GABA sensors) Calcium sensors The latest calcium sensors from Janelia (jGCaMP7) continue...be used for imaging small neuronal processes (dendrites and axons). jGCaMP7c exhibits high contrast between...

...contain a variety of restriction enzyme cut sites (restriction sites) downstream of the promoter as part of...these stains see the Bitesize Bio Blog and their associated manufacturers websites. These stains require...and insert - these both must have compatible cut sites for restriction enzymes that allow your insert to... a multiple cloning site (MCS). Having multiple sites allows you to easily orient your gene insert with...promoter (blue arrow) followed by the restriction sites EcoRI, XhoI, and HindIII. To place your gene in ...flanked on both sides by that enzyme’s restriction sites, but the insert can then anneal to the backbone ...digest large amounts of DNA in as little as 10 minutes, but check with your enzyme’s manufacturer to ensure...

... 20’s, new graduates often ask me for advice on careers available to new science graduates and the pro...better. I worked in a Microbiology testing facility, where I tested materials for bacterial contamination... in Spanish or French, and so they were ideal candidates for these positions.  I had to reassess what ...Standard Operating Procedures (SOP’s) and every item I tested was constantly tracked with barcodes and spreadsheets...the constant fear of failure because I knew the tests worked and would produce a reliable result. This...quickly; as soon as I learned how to do the different tests and became efficient, I then started to become bored...Molecular Genetics to give me an edge over other graduates during the recession. As each advertised job received...

...reasons, it is essential to test the selectivity of your antibody. But testing is not one size fits all....important to test for the protocol you intend to use in your experiment. An antibody that demonstrates selectivity...best to determine this by testing them. For intracellular targets, we usually test 3 different fix/perms ... Correctly interpreting your data therefore necessitates careful design of RNAi sequences, confirmation...like a blood sample containing many different leukocytes. Where data is available from antibody-independent...quite useful. HCDM, among other things, works to test flow cytometry antibodies through their HLDA workshops... labeling patterns on different primary blood leukocytes. The file shared also contains information about...

...important for candidates during a job search and it is even more critical for international candidates. Being...international candidates that they should join their relevant associations/groups. These candidates should also...looking to continue their careers in the United States. There has been a growing shift away from employers...with filing a permanent residence case with United States Citizenship and Immigration Services (USCIS). If...years to work (up to 40 years for recent Ph.D. graduates). Therefore, it is imperative to plan for and ...following paths to maintaining a status in the United States including green cards that are well-tailored to...startups and firms with a non-international focus. Candidates in need of an industry H-1B need to be aware ...

...following a report that one can use linear repair templates (ie. PCR-derived dsDNA) with 30-60 basepair homology...ribonucleoprotein complex with linear DNA as repair templates which reduces time to generate edits to only four...rescued F1 animals (gel image under worm). Image courtesy of Jordan Ward, UCSF. In 2015 (Ward, 2015)...optimize a range of editing parameters: oligo repair templates with homology arms of 35-80 bp, and DNA double-strand...guidelines for designing single-stranded repair templates (Farboud et al., 2019).   The Mello lab also reported...of oligos used to generate linear dsDNA repair templates by PCR boosted efficiency (Ghanta et al., 2018...that melting and rapidly cooling dsDNA repair templates dramatically boosted editing efficiency (Ghanta...

...editor had relatively high indel rates ranging from 4.7% to 30% at tested sites. Still, it opened a new avenue...abasic sites. This approach resulted in a maximum A → T product purity of 66%. Pro tip! Abasic sites are ...also called apurinic or apyrimidinic sites and abbreviated as AP sites. A collaborative team from the David...retained considerable A → G editing activity at most sites, in addition to A → C and A → T activity. To drive... produce a lot of indels (up to 40% at one site tested with AYBEv3!). Although AYBE, AXBE, and ACBE do...UNG enzyme was modified to recognize T and/or C substrates. The HuidaGene Therapeutics team has deposited...of which acronym is which. Acronym Full name Notes   Base transition Base change between purines ...

...were designed to target genomic sites with no annotated function, i.e. sites that lack open chromatin marks...20,500 protein-coding genes. Unique, non-overlapping sites in the genome were targeted and predicted on-target...binding site, or a coding region. Targeting these sites should control for the effects of guide expression... 3) safe-targeting guides, which target genomic sites with no annotated function. In the growth screen... safe-targeting guides were depleted at greater rates than their non-targeting counterparts, suggesting...screen Next, the toxicity of guides with off-target sites was profiled. Guides with exact or 1-bp mismatch...off-targets were only toxic if they had 5+ off-target sites. Results from the screen also reproduced several...

...profile) and can also be used as a paternity test. The test is accurate, precise, robust, and STR profiles...for a range of testing, from animal doping to forensics, DNA sequencing to drug testing. You can find ...favour of biochemical assays followed by in vivo testing. The mantra being “you can’t trust those whacky...their origins. This technique is still used today to test for HeLa contamination. HeLa contamination is still...closely followed by T24 bladder cancer cells. Estimates of the amount of money wasted in basic oncological...Down-Regulation of HIF-1α by NDRG2 Expression Attenuates Proliferation and Invasion in Renal Cancer Cells...directly isolated from patients. In these cases, a test known as Short Tandem Repeat (STR) profiling can...

...2023. DNA damage drives genome instability and contributes to cancer, premature aging, and other harmful...displacement loop (D loop). The recombination intermediates can then resolve to complete the DNA repair ...a one-ended DSB). This single invasive strand initiates primary synthesis from a template followed by ...sequence to be modified. In general, the insertion sites of the modification should be very close to the ...determinant when selecting a type of donor. ssDNA templates (referred to as ssODNs (oligodeoxynucleotides)...insertions such as fluorescent proteins or selection cassettes. This class of template should have homology arms...1000 bps. HDR efficiency with circular plasmid templates is generally low; to increase the frequency of...

... the backbone vector has multiple BbvCI sites. If these sites have opposite orientations, then both strands...the sites are in opposite directions, you just need to use a plasmid with BbvCI restriction sites in the...Scientists use deep mutational scanning to simultaneously test how multiple amino acid changes affect a protein...Hundreds of thousands of protein variants can be tested in one experiment. There are many functional assays...choosing and validating a selection system. You can test the effect of multiple mutations in combination ...shuffling to combine pairs of mutations together to test combinatory effects. One-pot saturation mutagenesis...comprehensive mutagenesis library that’s ready to be tested in a functional screen of your liking. This method...

...tip! Abasic sites are also called apurinic or apyrimidinic sites and abbreviated as AP sites. To explore...efficiency of up to 68% at the most favorable target sites. Differential cytosine editing between bacteria ...reliance on the glycosylase UNG to crease abasic sites (Zhao et al., 2021). Their GBE produced C → G edits...with over 50% efficiency in 23 out of 30 target sites. But the same strategy in E. coli produced different...bases — but E. coli instead filled those abasic sites with A's, producing C → A edits. Figure 3...base editors, so the Yidi Sun and Erwei Zuo labs tested and implemented several of these advances for CGBEs...HMCES forms a chemical crosslink with DNA abasic sites, protecting them from cellular processes that would...

...steadily increasing. Of course, you want the ‘latest and greatest’ variants with the best properties for your... affinity and a tendency to form higher order aggregates. This property can be tolerated in some applications...these experiments are discussed here. In vitro tests for aggregation The tendency of purified fluorescent...dimerization tendency. Another technique that separates molecules based on size is ultracentrifugation...of homodimerization in aqueous solution. In cyto tests for aggregation To what extent does homodimerization...cellular assays have been proposed. Early efforts tested whether or not FP-tubulin fusion proteins could...form microtubule networks in cells. Other efforts tested whether FP fusions to connexin localized correctly...

... junction proteins cx43 GJB1 GJB2 Bioelectric states can alter cell types and patterning There is mounting... or cancerous it is (Levin et al., 2017). These states can even be necessary and sufficient for certain... the entire worm during regeneration, the worm creates two heads. Subsequent cutting results in a two-...visualize or perturb these slower-changing bioelectric states in living cells. Since bioelectrical signals are...indicators (GEVI) are ideal tools to track bioelectric states (Matthew et al., 2018). These allow for not only...only recording but also modulating bioelectric states in vivo. For example, the Levin lab from Tufts University...fusion of the voltage sensing domain of a Ciona intestinalis voltage sensor and a mutated version of the ...

...fragments. When designed correctly, the recognition sites do not appear in the final construct, allowing for... the gene of interest is designed with Type IIS sites (such as BsaI or BbsI), that are located on the ...outside of the cleavage site. As a result, these sites are eliminated by digestion/ligation and do not ...final construct. The destination vector contains sites with complementary overhangs that direct assembly...transient construct retains functional Type IIS sites and will be re-digested. In contrast, formation ...construct does not retain the enzyme recognition sites. As a result, the ligation process is close to 100%...of fragments, and the loss of enzyme recognition sites after ligation favors formation of the construct...

... lab built synthetic promoters from scratch and tested to see if they drove cell type specific expression...that are upstream of the transcription initiation sites of at least two genes with high expression and high...cell type specific transcription factor binding sites interspaced with random sequences.   Strategy #4...AAVs were first injected into the eyes of mice to test for cell type-specific expression of GFP. Half (...promoters that are also unlikely to express in primates and humans. On the other hand, the ability of ...cell types both promoters expressed in. The lab tested this application using one promoter to drive expression...experiments: While synthetic promoters with high rates of labeling are ideal of gene therapy applications...

...glycoprotein (BaEVRless) had high rates of transduction for most cell types they tested. Interested in using Nanoblades...technologies, Nanoblades had higher rates for editing efficiency and lower rates of off-target effects. Nanoblades... mouse bone marrow (BM) cells, primary human hepatocytes, and hematopoietic stem cells (HSCs) using Nanoblades...from GFP transgenic mouse  GFP  ~60-65% Human hepatocytes  Myd88  ~50% Human HSCs  Myd88  ~50% Table...dsDNA donor template. In vivo editing of mouse zygotes and mice with Nanoblades CRISPR has also been extensively...used to create transgenic mice by microinjecting zygotes with CRISPR components. However, this sometimes... to disrupt the tyrosinase (Tyr) gene in mouse zygotes, which results in albinism in mice. Of the eight...