...when studying them since their complete knockdown is lethal. Incomplete or titratable knockdown, however...generations. Figure 1: Mobile-CRISPRi's modular components. Image from Peters et al., 2019 with permission... Top: A Tn7 transposon containing the CRISPRi components and a plasmid with the Tn7 transposition genes...ability to synthesize diaminopimelic acid (DAP), a component of the cell wall. Once in the recipient cell, ...Bottom: An ICE element containing the CRISPRi components is transferred to recipient bacteria by bi-parental...was variable. They measured transfer rates by comparing the number of colonies that grew on a selective... gRNA were more sensitive to trimethoprim when compared to control gRNA treated or untreated bacteria....

...antibody-conjugated labeling. Even though the two compounds are completely different in nature (eGFP being a protein...results in a higher-wavelength emission spectrum compared to APC. This happens due to a process called Fluorescence... red laser excites at 640 nm (Table 1), while comparing the two fluorophore's excitation spectra around...think of brightness levels on your smartphone or computer screen), and you should be able to properly label...the same color panel. In this case, make sure to compare the excitation/emission spectra of your reporter...whether and to what extent they overlap. Figure 4 compares the spectra of enhanced GFP (eGFP) and FITC, the...Make sure to check out our blog post on color compensation to learn more about them. Alright, let's build...

...referred to as “complex retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, ...biology. “Retroviruses” is an umbrella term which encompasses 1.) the gamma-retroviruses — that have been developed...whole family; however, you can use Figure 1 to compare the genomes of these two family members.   ...beginning to realize that the situation is already complex enough that a deep understanding of retroviral ...Because it is made at the same time as the other components of the vector particle, some of the RT protein...In both cases, the lipid membrane is the only component that is not dependent on viral genes. It is derived...Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiological Reviews, 56(3), 375...

... if you can begin simply and build up to more complexity if needed. And since we live in the digital age... biology and ecology can make for beautifully compelling photos and videos, while molecular, micro, and.... Can you convey enough information to tell a compelling story in an image or do you need a full exhibit...fair, or a semester-long after school program? Comparing your desired outcome to the limitations and potential...expertise needed can increase exponentially with complexity. Podcasts Medium Easy for audience to engage... needed. Traditional media formats are quite competitive while digital can be oversaturated. Transition...Photography Medium (high for microscopy) Visually compelling. Easy to convey a large number of details without...

...of the vector components are HIV-based? A7: No single plasmid contains all the components necessary to ...The components are divided as follows: Transfer vectors contain minimal cis-acting HIV components: LTRs...decreasing the probability of creating replication-competent lentivirus (RCL) in your target cells.  Q4: How...targeted. Q6: Are transfer plasmids replication competent? What is SIN? A6: Most (if not all) Addgene transfer...LTRs, PPT, RRE, and psi packaging signal. Viral components typically total <1.5kb, which is rarely more ...are: 1) the potential generation of replication competent virus [usually HIV-1]; and 2) the potential for...

...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...

...  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...

...intense amount of competition between microorganisms for available resources. This competition drives microbes...antibiotic compounds. In such a case, the microbe itself would be unharmed by the compound it produces...treat infections, we are treating microbes with compounds similar to those they have been battling for centuries...treatment regiment might wipe out bacteria that are completely susceptible to a drug while a full course of ...likely to develop over time. It isn’t feasible to completely eliminate the development of resistance; it’s...hygiene standards. Handwashing is an essential component of preventing infection, as is cleaning and disinfecting...

...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...

... 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...

...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...

...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...

...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...

...derived from AAV9, their efficacy is generally compared to the AAV9 serotype or an engineered precursor...doesn’t lead to enhanced transgene expression, compared to AAV9, in BALB/cJ mice but is more effective..."Mouse Strain-Specific Tropism?" column. For a complete list of mice strains with LY6A-dependent tropism... by the Gradinaru lab (Deverman et al., 2016). Compared to AAV9, it effectively transduces neurons and...toward astrocytes, but its neuron transduction encompasses more molecularly-defined neuronal subtypes and...astrocytes and neurons, with greatly increased efficacy compared to AAV9 (Hanlon et al., 2019). This capsid was...brain regions, with reduced off-target expression compared to AAV9 in infant rhesus macaques. The AAV.PAL...

...multiplex (Thellwell, 2000). See Table 1 for a comparison of features of several commonly used chemistries... multiple reference genes whenever possible (Vandesompele, 2002), and it's possible to multiplex your ...documented in the literature, a geNorm study (Vandesompele, 2002) will help you select the appropriate ...have pipetting errors on the edges of the plate. Compare the CT values within each set of triplicates to...reaction, each target in the reaction should be compared to an individually run sample when developing ...value representing the fold change of each sample compared to your control. But as we said previously, just... See Table 2, (adapted from Wong, 2018) for a comparison of different analysis methods.   Method ...

...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...

...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...

...binary systems. In recent years, a number of more complex systems have also been developed which don’t fit... Bujard, both the promoter and transactivator components of the system have been engineered and improved...revolutionized genetic studies in flies. An additional component, the GAL80 repressor, adds a repressible element...UAS-driven gene expression. To further increase the complexity of the system, researchers have used split GAL4... developed by Lai and Lee. LexA/lexAop is a complementary system to GAL4/UAS that functions in essentially... cells and ~24,000-fold in human HeLa cells as compared to basal expression. Co-expression of repressor...because it allows them to be used together for complex genetic analysis in Drosophila or for precise control...

...Using AAV vectors in complex biological systems can be tricky at best, and downright infuriating at worst...Serotypes Serotype refers to the different protein compositions of AAV capsids, which determine how a virus ...what works great in one system or cell type can completely fail in a different system (Issa et al., 2023...designing and testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two ...Taschenberger et al., 2017).     Figure 3: Comparison of two astrocyte selective promoters demonstrating...Konishi, Y., Hosokawa, Y., & Konishi, H. (2014). A comparative analysis of constitutive promoters located in...vector-mediated transduction of the corticospinal tract: Comparison of four promoters. Gene Therapy, 28(1–2), 56–...