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...sequencing or restriction digest! Then, you can transform or transfect the cells that you want to use for your...project, scientists are no longer limited to traditional restriction enzyme cloning. Instead, you can ... reactions are performed, the next step is to transform competent E. coli cells and select the positive...expression clone. You will also need to use a E. coli strain sensitive to CcdB (e.g. DH5α, TOP10, Mach1). The... inhibits the growth of CcdB sensitive E. coli strains, most colonies should contain the desired, recombined...of the KRAS coding sequence. If you choose this strategy, it’s important to include the proper protein ...reading frame considerations, etc). This video demonstrates how to use the Snapgene program to design Gateway...

...expression of a transgene (Lin et al., 2018). However, this system requires precise titration of the AAV. ...visualize individual/non-overlapping cells. While transgenic animals are commonly used to specifically manipulate...are affected. Previous tools to overcome this restraint include an AAV-based sparse labeling system where...tools in Drosophila (Fig. 1):  GAL4-UAS, a transcription factor binding site. GAL4 expressed in the fly...site to cleave and inactivate any read-through transcripts of the effector gene. Effector genes, any gene...cassette remains following recombination. This strategy results in sparse labeling of the cell population...across cell types and organisms by replacing the transcription factor binding site and effector in the construct...

...selection strategy, here are a few to consider. Antibiotic selection Selection is straightforward in cases...re-ligated back on themselves. Therefore, your cloning strategy needs to identify cells containing the plasmid...includes an antibiotic resistance marker. After transformation, plate the cells on medium containing the corresponding...selection will select for cells that have been transformed with the plasmid backbone (with or without your...ve designed your experiment using an auxotrophic strain of bacteria (one that doesn’t synthesize an essential... the critical amino acid). Many common E. coli strains used for cloning are auxotrophic for specific amino...Toxin-antitoxin systems One negative selection strategy uses the toxin CcdB. Gateway cloning takes advantage...

...). If you can get away with generating transiently transfected cells, you will not need a mammalian selection...design. You can create a new strain with a disrupted gene by transforming wild-type yeast with an integrating...whistles to your construct. If you are planning to track your protein in the cell or purify it – and you ...mammalian selection cassette to screen for positive transfected cells. The addition of an in-frame fluorescent...plasmid shuttle vectors. You may prefer the more traditional approach of an autonomously replicating vector...a wild-type version of YGOI to rescue a mutated strain or a mutated version of YGOI to test the different...applications used to study gene function. From making transgenic animals, gene targeting and genome modifications...

...to the lysosome, and degradation of autophagic substrates inside the lysosome. Historically, methods for...autophagy, the untargeted Rosella remains in the neutral pH cytoplasm where it emits red and green fluorescence...mitochondria-targeting sequence, Rosella can be used to track the targeted autophagy of mitochondria, or mitophagy...Rosella can also be fused to a protein of interest to track the autophagy of that protein. Find the cytosol-targeted...proteases is key to its pH-dependent fluorescence. In neutral environments, dimeric Keima is excited at 440 nm...mRFP-GFP-LC3 Tandem Fluorescent Protein, but following translation, this fusion protein is cleaved to generate one...140.3 (2010): 313-326. PubMed PMID: 20144757. PubMedCentral PMCID: PMC2852113. Noda, Takeshi, and Daniel...

...lineage. Variations of this library allow tracking of transcription or chromatin accessibility at the single-cell...Who doesn’t love a library? A traditional library might contain thousands or millions of different books...libraries Just as physical barcodes help librarians track individual books, genetic barcodes allow researchers...descendants. This makes them useful for lineage tracing, among other applications. Cells can be identified... however, nuclease-dead Cas9 is fused to a transcriptional activator or repressor to create a reversible...assessment library, and a prime editing lineage tracing library. Other screening libraries CRISPR isn’t...Zhang lab developed this library of all human transcription factors, including splice isoforms, to allow...

...com­plex is very efficient at introducing double stranded breaks (DSBs) into genomic DNA in many cell types...homologous DNA repair template, such as a sin­gle-stranded oligodeoxynucleotide (ssODN), which contains the...repaired by NHEJ. While other groups have focused on strategies to improve overall HDR rates, we have shown recently...developed a framework that, by combining these two strategies allows you to specifically incorporate homo- ...the cells. However, our excitement turned into frustration when we sequenced our cells and found how inefficiently...indels. Following this observation, we tested a strategy that was discussed in the field at the time but...to change the PAM site, however we have also demonstrated the efficiency of guide RNA blocking mutations...

...experiment (i.e. the spectral properties match your available imaging instruments and the spectral properties do...allowing one to “directly” visualize their target. In contrast, indirect IF requires two antibodies: a primary... larger collection of primary antibodies. This strategy gives you the flexibility to target primaries ...relate to one another in the same sample. For this strategy to work, it is important to ensure that you are...secondaries with fluorophores whose excitation/emission spectra do not overlap. Multiplexed IF can also require...need to optimize the type of block used, the concentration, or the incubation time depending on your target...target molecule. You may need to optimize the concentration and incubation time. Secondary Antibody ...

... while the intracellular T cell receptor portion of the CAR facilitates signal transduction and release...useful for imaging applications; better tissue penetration which is useful for therapeutic and imaging applications... you generate an scFv?  scFv sequences have traditionally been cloned by amplifying the VLand VH antibody...proper disulfide bond formation.  expression in strains of bacteria that are redox mutants so they have... has led to their application for basic and translational research.  Basic research applications of scFvs... signal is boosted and enables single molecule tracking in living cells. The HA frankenbody is an HA tag...generate since it’s genetically encoded.   Translational applications of scFvs: Just like antibodies...

...2015, there were six of us. We were initially strangers but we all shared a passion for DIY Biology and...December 2015 we held our first intro to bacterial transformation course (read our blog post on it and to see...consisting of over twenty members and volunteers. Transforming citizens into scientists Anyone can become a...mathematics, and more. That’s right, we have non-traditional scientists working in our laboratory! Don’t worry...that everyone gets proper equipment and safety training prior to getting started. For example, Timothy...dedicated time at BosLab, he became our bacterial transformation guru and instructor for our intro to biohacking...BosLab. Once he optimizes plant cell culture and transgenesis he wants to share his knowledge with the community...

...develop more transferable skills? See Developing Transferable Skills During Science Training and So You ...British Columbia. Originally on track for a research career, she transitioned into a communications role instead...of the University of British Columbia. When transitioning from an academic science career path to a non-academic...advantage over other candidates. Write about your transferable skills Some of the skills you developed might...of that, perhaps you were involved in some extracurricular activities – did you volunteer to do science...have presented in some public lectures? These transferable skills could be meaningful for your potential...

...alone. A few years ago when I started my career transition from bench science to software product management... a strong background of undergraduate research straight to a lab career at a biotech company, I always...explaining more of her role, which eventually led me to transfer to her team and begin my product management (PM... I want to be able to share more about this nontraditional career path for others who love science, but...science backgrounds, most did not. It was my job to translate complex lab operations workflows into a format... help improve their work. Then, I distill and translate that valuable information back to the rest of ...science companies, try to find internships with translatable skills: working with data, working with people...

...the delivered cargo (often a gene) is called the transfer plasmid while the other plasmids with packaging... or something similar, and is contained on the transfer plasmid. If expression is the goal, a promoter...The genetic cargo requires viral packaging to infiltrate and integrate into the host genome. First, it...Pol, the last component, contains the reverse transcriptase and integrase components to convert the cargo...gamma-retrovirus, but they perform very similar functions to infiltrate the host cell and deposit viral cargo. Gene expression...other to allow DNA synthesis of the complementary strand to yield dsDNA. Unlike LTRs, ITRs are perfectly...delivered and expressed in the host cell from the transfer plasmid. Viral packaging and entry The viral packaging...

...number during the transition to stationary phase, Segall-Shapiro et al. placed a trans-acting RepA protein... the plasmid by altering IPTG concentrations. Higher IPTG concentration leads to higher RepA expression... the team made use of stabilized promoters. In contrast to unregulated promoters that are always on, stabilized...because TALEs can be programmed to bind tightly to arbitrary DNA sequences and have been shown to achieve ~...high plasmid copy number. When they increased concentrations of IPTG, a constitutive promoter upstream of...optimization of gene dosage in E. coli using DIAL strains." Journal of biological engineering 5.1 (2011):...): 10. PubMed PMID: 21787416. PubMed Central PMCID: PMC3163176. Resources on the Addgene Blog Read the...

...and IREB2, which post-transcriptionally regulate iron metabolism pathway transcripts. By using RaPID, Ramanathan... include both scrambled RNA motifs and cells transfected with BirA* protein alone (i.e. no RNA bait). ...experiments. These background proteins were then subtracted from Ramanathan et al.’s RaPID datasets. Applications...changes in RC2H1 protein expression and post-transcriptional regulation of SM1v1-containing RNAs could play...often trick host proteins into binding their untranslated regions (UTRs) to aid in the viral life cycle...IRE) RNA motif of the ferritin light chain RNA transcript. IRE motifs are bound by IRE binding proteins...Methods, 15, 207-212. PubMed PMID: 29400715. PubMed Central PMCID: PMC5886736. Find the RaPID Plasmids Here...

...they are not always easy to track at the transcriptional or translational level. However, several genetic... 20026026. PubMed Central PMCID: PMC2823934. Pai, Vaibhav P., et al. "Transmembrane voltage potential ...fluorescent voltage indicators (GEVI) are ideal tools to track bioelectric states (Matthew et al., 2018). These...Use of genetically encoded, light-gated ion translocators to control tumorigenesis." Oncotarget 7.15 (... (2016): 19575. PubMed PMID: 26988909. PubMed Central PMCID: PMC4991402. Levin, Michael, Giovanni Pezzulo...(2012): 313-323. PubMed PMID: 22159581. PubMed Central PMCID: PMC3243095. Additional resources on the ...

...surface display vectors to improve expression and trafficking of these biosensors.   Figure 1: Schematic...hippocampal cultured neurons, imaged 14 days post-transfection, excited at 505 nm (right). Scalebar represents...hydropersulfide-propagating enzymes 3-mercaptopyruvate sulfurtransferase (MPST) which catalyzes the desulfuration of...an enzyme-bound hydropersulfide that is then transferred to thiol-containing acceptors. These hydropersulfides...redox modification, the MPST hydrosulfide can be transferred to the roGFP2 cysteine thiols and change its ... human cell line, Hep3B. From left to right: Mitotracker Red, GFP for the biosensor, Hoechst, and overlay...signaling related fluorescence during morphine administration and withdrawal to explore how these cells may...

...students transform them into different strains of E. coli,” says Westenberg. During the transformation experiment...different genetic backgrounds of the host strains affect transformation efficiency.  Aside from the Streak Plate...add a bit of fun. He mixed together 10 E. coli strains producing different pigments, and tasked the students...students to separate out colonies from each individual strain using the streak plate technique. Time for the...single colonies, these chromoprotein-expressing strains are easily amenable to other experiments to teach...

...Interactions Difficult to detect weak and/or transient PPIs. Many PPI identification techniques rely ...often prevents the identification of weak and/or transient interacting proteins. No spatiotemporal control...BioID or Split-BioID, cells are lysed, protein extracted, and labeled protein interactors purified via ...BioID, is also useful for identifying weak and/or transient protein-protein interactions since the biotin ...speed of labeling. BioID requires 6-24 hours for tetracycline induced expression of the BirA* fusion protein...Split-BioID, Schopp et al allowed 24 hours for tetracycline induced expression and biotinylation of interacting... communications. PubMed PMID: 28585547. PubMed Central PMCID: 5467174. Find the Split-BioID Plasmids ...

...the academic science landscape: the treatment of trainee scientists as workers, at least for the purposes...purposes of labor laws, rather than as trainees in a special category, as has been the ‘labor-of-love’ philosophy... on long-term efforts to better account for and track postdocs nationwide. On this level, FoR is currently... by the development and implementation of new strategies designed to cope with current increases. These...will include a discussion on effective advocacy strategies on various levels, with panelists including: ...effectively advocating for change: from developing a strategy to distilling a message for the appropriate audience...2014): 5773-5777. Pubmed PMID: 24733905. Pubmed Central PMCID: PMC4000813. 2. Taylor, Justin Q., et al....