... cellular behaviors requires making complex manipulations to cells. For example: in a cell there are proteins...other proteins and with DNA, and these complexes regulate the transcription of specific genes. What if one... one of those genes coded for a protein that regulated its own transcription, or the transcription of ...These interactions yield a cellular phenotype, translational output, etc., which can support a larger cellular... is made, modified (sometimes many times), and isolated afterwards. To engineer these production pathways... them to these organisms via another optimized platform. Gene Stacking As the name implies, gene stacking...identify the best cocktail of genes when the relationship between genotype and phenotype is best described...

...VP1, which self-assembles into a compartment encapsulating the protein of interest (Figure 3).   ...D-glucaric acid biosynthesis. This MPyV system encapsulated a rate-limiting enzyme in this pathway (myo-... of the protein compartment. Yeast with this encapsulated enzyme produced 20% more D-glucaric acid, suggesting...system can improve metabolic pathway yields by encapsulating a single enzyme. Compared with previous research...captures rare clones that represent 0.001% of the population.  Because indel generation by Cas9 can occur ...steady rate, scientists found a way to use the accumulation of indels to measure time. These plasmids with...ready-to-use viral preps with more on the way. S5E2 regulatory element containing vectors developed in the Jordane...

...liquid media or solid media (on plates). In general, amplification on plates can help to ensure that diversity... anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the library, but generally needs...transformation efficiency, or more specifically calculated based on the size of and weight of your library...library varies significantly in plasmid size). On plates, each transformed bacteria will be its own colony...Screening Zhang Lab - Multiplexed Overexpression of Regulatory Factors (MORF) Library 3,548 250 - 10,000...Screening Rinehart Lab Iterative Synthetically Phosphorylated Isomers (iSPI) Libraries ~11,000–110,139 ...

...individually generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it..., as well as the 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There is no need for PCR or ...the second site accepts the homology arm repair template. SapTrap includes a prebuilt donor plasmid library...be used to generate functionally unique repair templates. A final ligation reaction correctly assembles...genetic locus, the toolkit also offers repair templates for tagging proteins in a tissue specific manner...of auxin to the culture medium. Any number of regulatory sequences, including protein enhancers and repressors...repressors, can be included in the repair template for inducible alteration of protein expression levels...

...that grew on a selective plate versus the number that grew on a non-selective plate. Most bacteria tested... types of bacteria: lab-adapted, environmental isolated, and disease-associated. Mobile-CRISPRi: a modular...glmS gene in the recipient's genome. Antibiotic plates lacking DAP select against donor bacteria and for...including pathogenic strains and environmental isolates. For example, Peters and colleagues used Mobile-CRISPRi...aeruginosa) in addition to Vicrio casei, a bacteria isolated from cheese. Knockdown efficiency, as measured... 100-fold excess of wild-type bacteria and the relative frequency of each of the 40 library strains was...

...homologous recombination using a repair template. These templates can include loxP sites to create conditional...recombination. The concept is simple: deliver a template with a modified version of the targeted sequence...sequence to the cell which will recombine the template with the endogenous gene. Here, we'll describe the ... one of the homology arms. When we select cells later, we will first perform a positive selection for ...selection for the negative selection marker - this latter step will kill off many of the cells that have ...knockout targeting vector designed to insert and later remove a resistance gene. In this construct, the...

...exciting directions. Drosophila are very easy to manipulate genetically and have limited genetic redundancy...faster but are also more stressed by the heat. Isolating male and female flies Differentiating male and...make genetic crosses properly, it is important to isolate females from males while they are still virgins...genomes Compared to humans and mice, flies have a relatively simple genome. They have four chromosomes: the...are miniscule, so researchers generally do not manipulate them. Essentially, the X, second, and third chromosomes... phenotypes, check out the Classical Genetics Simulator website. One interesting thing to know is that... systems. Some resources will direct you to the latest information about gene function, while others offer...

...screening experiment is to generate and screen a population of mutant cells to identify genes involved in...experiments commonly use lentivirus to deliver a pooled population of gRNAs to Cas9 expressing cells. Pooled lentiviral...CRISPR libraries”) consist of a heterogeneous population of gRNA-containing lentiviral transfer vectors...the CRISPR library to generate a heterogeneous population of mutant cells, with each cell or set of cells...distribution is analyzed in the drug-resistant population compared to a non-treated control group. In this...maintaining the distribution of each gRNA within the population is key. Loss of representation due to enrichment... sufficient representation within your mutant population requires a massive amount of cells as starting...

... for screening protein-protein interactions. In later years, improvements to yeast transformation protocols...can either be applied to the bait in a single population (e.g. transforming both fusion plasmids, or mating... strain for screening), or arrayed on a 96 well plate where each well contains a defined prey fusion.  ...former allows for screening greater numbers, but the latter eliminates the step of identifying prey proteins...protein expression and correct folding, post-translational modification, protein degradation, access to...problem is that your proteins are not post-translationally modified, co-expression of the enzyme responsible...transcription-activating function of a eukaryotic regulatory protein. Science. 1986; 231:699-704. PubMed PMID...

... to copy and modify templates (e.g. http://openwetware.org/wiki/Protocols/Template). Nature Protocol Exchange... up after trying out the new protocols.io beta platform for sharing, annotating, and storing life science...molecular cloning protocols, including making LB agar plates, restriction digest of plasmid DNA, and bacterial...bacterial transformation. And more plasmid-related educational resources can be found in our Plasmids 101 blog...creating and editing Wikipedia pages, OWW will be relatively easy for you to begin using. For the novice, ...the repository. These protocols show up on the related plasmid pages and can help scientists use these...

...in immune cells have identified many putative regulators of immunity. However, in vitro screens cannot... a Thy1.1 selectable marker to facilitate the isolation of modified cells; and (5) is compatible with ...conditions for a screen that assessed genes that regulate antigen-specific CD8+ T cell responses to a murine...responses to cancer. The screen recovered known regulators, such as Ptpn2, Pdcd1, and Zc3h12a, demonstrating...Moreover, the screen identified putative novel regulators of CD8+ T cell responses in tumors, demonstrating...Cancer and the Immune System: Deciphering the Relationship...

...Next-generation sequencing is performed on viral genomes isolated from the final AAV preparation. Sequencing results...of AAV preparations is assayed by comparing the relative stoichiometric ratios of the viral capsid proteins...SYPRO Ruby staining and the molecular weight and relative intensity of the viral capsid proteins are analyzed...Viral capsid proteins VP1, VP2, and VP3 are shown relative to the total protein present in the sample. ...are added to cells in culture. Three to five days later, cell cultures are inspected for sterility. Transducibility...-AAVrg) alone at 1.1E6 vg/mL, or both. Two days later, Cre-dependent GFP expression was detected with ...vectors consist of full particles (white arrowhead) relative to empty particles (black arrowhead). Scale bar...

...within the cell. Most plasmids encode a negative regulation system involving antisense RNAs or iterons that...indirectly inhibit replication through inhibiting translation of replication machinery proteins, or directly...transcription is repressed and replication is downregulated. In the second model, termed the “handcuff” ...cases of compatible plasmids, different negative regulation systems control plasmid replication of each plasmid... the same origin of replication, the negative regulation system cannot distinguish between the different...different plasmids. The concentration of negative regulation system elements in the cell is artificially high...). However, more recent studies suggest that the late replication that occurs for some types low copy ...

...dCas13a may be useful for isolating specific RNA sequences from a population (either enriching or depleting... proteins for imaging, tracking, modulating splicing, and regulating expression of specifically targeted...Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but they require both a tracrRNA and crRNA...the spread of infection throughout the entire population. This property of Cas13a opens up the possibility...knockdown to single gRNA expression. Although the collateral RNA cleavage response is well described in bacterial...

...been battling for centuries. As such, resistant populations can arise very quickly following the tried and...showing bacteria evolve antibiotic resistance on a plate! Antibiotic resistance can arise, and has arisen...antibiotics These scenarios expose bacterial populations to antibiotics at unnecessary times, putting ...pressure on resistant bacteria to over-take the population and making them more difficult to treat in the...susceptible bacteria that will be more difficult to treat later. A similar situation can occur if you treat a bacterial...because scientists in the laboratory will need to isolate and grow whatever bacteria are causing the infection...before they can test for resistance. Growing and isolating the infecting bacteria can take 24 hours or more...

...transcriptional activators. These constructs allow for the upregulation of essentially any gene by fusing mutants of...cause differentiation and to reverse HIV viral latency (Bialek et al., 2016). Additionally, CRISPR-based...factors that may prevent your gene from being up-regulated. For instance, generally, the more highly expressed...Additionaly, cDNAs cannot be used to study in cis regulation and also suffer from an inability to easily deliver...CRISPR-based tools for altering DNA sequence or regulating gene expression.     References Behan FM, Iorio...Stucka R, Hauber J, Lange UC (2016) Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional...for inducible genome editing and transcription modulation. Nat Biotechnol 33:139–142 . https://doi.org/...

...sessions, exhibit booths, happy hour socials, and late, late night socials. As a newbie to the SfN conference... global coffee supply… My back-of-the-napkin calculations put the daily consumption here at ~ 60,000 cups...students and postdocs, which accounts for many, many lattes and espressos. The energy boost is well needed ... Investigator Awards and we couldn’t wait to congratulate him on his successes. We've been lucky enough...SfN14 on Twitter for live tweeting updates and the latest news from attendees. And for those of you presenting...

...have similarly curated their Ebola-related content. -And the latest updates on the ongoing outbreak from... underestimate. Given all the excitement and speculation, we thought we’d do a little digging and assemble...assemble a rough guide to the science of Ebola and related resources. Ebola Basics A few basics: Ebolavirus... data on five strains of Ebola representing the latest outbreak and earlier ones, along with links to ...the current Ebola crisis, Science and Science Translational Medicine have made their complete collection...

... target to specific subcellular regions, laser ablation can damage other cellular structures, and genetic...genetic mutations are not easily reversible. Late last year, the Akhmanova Lab published a new optogenetic...for a wide variety of experiments. Opto-RhoGEFs manipulate cell morphology and adhesion But what if you’... Taking control of a RhoGEF is a great way to stimulate and study these physical processes. Joachim Goedhart...of membrane-recruitment optogenetic tools for manipulating Rho GTPases based on BcLOV4. These single-component...membranes) (Figure 4). The authors used SuperPLD to manipulate phosphatidic acid signaling (such as Hippo, AMPK...minutes of blue light illumination, substantial PA accumulates in the plasma membranes, indicated by the PA ...

...often requiring several rounds of selection to isolate plants with the phenotype of interest. Genome engineering... modification of known or suspected genes that regulate a desired phenotype. In fact, CRISPR has already...variants have been created in an effort to increase translation in a particular plant species or cell type (Belhaj...driven by plant-derived RNA pol II promoters which regulate expression of longer RNAs (such as mRNAs for gene...plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies. New...Multiplexed Plant Genome Editing and Transcriptional Regulation. Plant Physiol 169:971–985 . https://doi.org/...(2016) Gene-edited CRISPR mushroom escapes US regulation. Nature 532:293–293 . https://doi.org/10.1038...