...  Here's what you'll find in this post:  New compact, efficient prime editors A versatile and precise...tape Teaching an old antibody new tricks   New compact, efficient prime editors By Susanna Stroik The ....), while PE6c and PE6d were most efficient at complex edits, such as long or highly structured templates...Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell, 186(18), 3983–4002...interchangeable set of fusogenic and targeting components to reprogram delivery vehicles like lentiviral...DIRECTED particles where the fusion and targeting components are interchangeable. Image from Strebinger et...the range of targetable cell types. DIRECTED is compatible with fusogens from different viral families and...

...the cell must have enough nutrients available to complete the entire round of replication. Initiation is...initiate replication. But pSC101 requires a third component: RepA, a protein encoded by pSC101 itself. RepA... are efficient for the host, as no additional components are necessary. Iterons are not the only tool ...Inc, also encoded by the ColIb-P9 plasmid, is complementary to part of the sequence forming this pseudoknot...restraining further replication.   Figure 3: Comparison of replication regulation mechanisms of stringently...replication. At high copy numbers, RepA proteins form complexes between iterons on different plasmids in a process...propagation by limiting their copy number and compensating for the additional metabolic cost imposed on...

...lab. Iron Chef-style competitions Every few months, we’ll have a cooking competition featuring a selected... eat together every day, sharing ideas for the company, but also talking about sports, movies, and just..., which is either an indication of the stiff competition or says something about my cooking.   This is... to organize. We’ll dedicate a post to these competitions in the future, and hopefully include some of...To-Do list long enough that there is no hope of accomplishing it all. How do we encourage people to take a... Hack Days, and we’ve seen great success at accomplishing “we-should-really-make-time-for-that” type projects...

...based on its library frequency before selection compared to its library frequency after selection. Key ...and easy technique that can be used to generate complex libraries of mutant plasmids ready for deep mutational...libraries, they all have inherent shortcomings compared to one pot saturation mutagenesis. Error-prone...vector with Gibson assembly. While this is a comprehensive mutagenesis method, a drawback to using Gibson...scanning libraries. PFunkel: Of prior existing comprehensive mutagenesis methods, PFunkel offers the best...second universal primer is used to synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled...PCR-based approach for generating a customizable comprehensive mutagenesis library that’s ready to be tested...

...likely not a coincidence that the AAV genome is composed of single stranded DNA, which is particularly ... interest, ordering them from a gene synthesis company such as IDT or Genscript, and cloning them sequentially...infection with adeno-cre. AAV-based gene editing compared to CRISPR editing CRISPR, of course, has started...and virtually never exceeds 40%. While this is comparable or better than the efficiency of most current...systems. Ohmori et al 2017 use a dual vector system composed of SaCas9 and a gRNA in one AAV and a repair template...researchers have devised methods to deliver CRISPR components as ribonucleoproteins (RNPs) that can be supplied...2017 recently showed that by delivering CRISPR components as RNPs and delivering two halves of a repair...

...now available. However, it can be difficult to compare their performance, as most translation initiation...directly engages the ribosome for translational complex assembly. PhD student Dejan Gagoski used SITS ...SITS to create the pCell-Free vectors, Gateway-compatible backbones that enable cell-free expression of proteins...-tagged human ORF clones to allow testing and comparison of different cell-free expression systems; the... construct barcoded, locus-specific libraries compatible with Illumina next generation sequencing in order...libraries using a 2-step PCR-based protocol and are compatible with single-end or paired-end read flow cells...21408-21413. PUF Assembly Kit PUF proteins are composed of repeat segments that can be engineered to bind...

... Now that you’ve completed a resource budget, and know how to decide which components are the most critical...an account of the material resources needed to accomplish your science, and can include: equipment, reagents...then start identifying the resources needed to accomplish that goal. 2. Identify the tools in that timeline...research success. If you can get away with using computer modeling and collaborations rather than using ... most start-up P.I.’s. It doesn’t have to be complicated, or difficult to create this list. Start BEFORE...equipment. That’s why a resource budget can help you compare the monetary and empirical value that helps you...from academia altogether and instead started a company around his own research. He was clear on his science...

...some clones being outcompeted by others in liquid culture, dilute and plate the complete library of transformants...faces as it approaches a host cell surface are complex polysaccharide structures, many of which are easily... DNA from host cell nucleases (4). To further complicate matters, even when sequenced, phage genomes tend... for cell-binding abilities (6). This can be accomplished using a restriction enzyme with a short recognition...involves forcing DNA through a small opening using compressed nitrogen gas; this shears DNA at random locations...your resultant plasmid libraries into chemically competent E. coli and analyze transformants for appropriately...transformation should be performed using a highly competent strain that you can use for long-term library ...

...to detect a single, specific protein within the complex mix derived from cells or tissues. Westerns are...sample per lane, onto the top of a resolving gel composed of the crosslinked polymer acrylamide. In addition...proteins are immobilized onto a membrane, usually composed of a nitrocellulose or polyvinylidene difluoride...membrane.  In a wet transfer system the sandwich is completely submerged in a tank filled with a transfer buffer...confirming that the protein is the expected size as compared to the protein standards.  Protein abundance In...loading control when assessing protein abundance or comparing samples across lanes.  For a typical loading control...present in all samples. Uneven sample loading or incomplete protein transfer causes inconsistent levels of...

...Burry 2011). Complex experiments Finally, additional controls may be required for more complex experiments...expected response and have a baseline with which to compare the experimental group.     Figure 1: A...western blot several times with the same samples and compare the results across runs. If the results vary considerably...will briefly touch on.  Western blotting When comparing samples in different lanes in a western blot, ... to the controls listed above, viability and compensation controls are recommended for flow cytometry....example, propidium iodide only stains dead cells). Compensation controls address the spectral overlap that occurs... occurs during multiplexing. Compensation controls are single stained samples for each fluorophore and...