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...Pick the right strain While common E. coli strains like DH5-alpha are recA-, some strains have been engineered...suggest gel extraction to isolate the full, intact plasmid. Please see our gel extraction protocol for...Learn about the common lab E. coli strains Find tips for viral transduction  ...Repeating sequences within a plasmid can undergo intramolecular DNA recombination. This naturally occurring...recombination, one resulting plasmid carries the transgene of interest (aka the viral genome) and the other...resistance gene and origin will replicate, and the transgene will be lost. Over time, bacteria containing the...further reduce recombination. Recombinase-deficient strains like Stbl2, Stbl3, and NEB stable E. coli reduce...

...biofactories by demonstrating the use of CRISPR-based RNA scaffolds to mimic natural transcriptional programs...recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds." Adam has...visualize the concepts discussed in the paper. The transcriptional control of multiple loci is deftly coordinated...cerevisiae with few techniques that are easily transferrable across host species or metabolic pathways. A...synthetic RNA scaffolds that can recruit various transcriptional regulators (Zalatan et al. 2015).  These scaffolds...nucleotide sequence then subsequently recruit a transcriptional regulator to modulate expression at that locus...expression levels dependent upon the type of transcriptional regulator or the number of designed scaffold...

...your strategy. I realized that similar types of thinking go into developing gene therapy strategies for...logic and strategy skills to apply those concepts and understand how therapeutic strategies work. Find...game. They had 35 minutes to develop a strategy and build a transfer plasmid that could be used to genetically... a win-win strategy. After each team presented, all students had to pick the best strategy and propose... This guest post was contributed by Marco Straccia, an Associate Professor at University of Barcelona...the University of Barcelona wanted to develop strategies to make our classes more practical and hands-...biology, microbiology, genetic engineering and strategic knowledge to solve a clinical disease case. As...

...:     Cells must be amenable to transfection or viral transduction     Appropriate promoters must be...expression     The requirement for Cas9 transcription and translation delays editing What are Cas9-gRNA ... studies in cells that are difficult to transfect or transduce. While using Cas9-gRNA RNPs may require...site of interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes... often be used in cells that are difficult to transfect, such as primary cells. Using RNPs can also alleviate... are detectable at high levels shortly after transfection, and are quickly cleared from the cell via protein...from Alex Schier’s Lab. gRNAs can be in vitro transcribed from ssDNA, which can be generated by commercial...

...Types of transfers Your transfer options are wet, semi-dry, or dry/electroblot. A wet transfer is the most... while semi-dry transfers require a semi-dry transfer cell. Dry/electroblot transfers only work in the...) steps to the transfer: you’ll need to pre-wet most PVDF membranes before the transfer and allow them...  Note that each type of transfer requires different equipment. Wet transfers can be done in an insert...strip/reprobe Semi-dry transfer Decreased efficiency Electroblot transfer Increased cost; decreased...strong as DTT, you’ll have to use different concentrations if you switch from one to the other.  Type ... 180 V, 30 minutes Good shelf life; runs at neutral pH (reduced protein alteration) Expensive buffers...

... by AAV? A: One potential strategy is to use split-inteins to mediate trans-protein splicing. Note, however...dose. The color diversity in Vector Assisted Spectral Tracing (VAST) experiments using PHP.eB in the brain...been able to successfully systemically transduce and then retransduce a model organism such as mice, e.g....problem? A: Re-administration is a challenge for AAV due to the development of neutralizing antibodies in... at a high MOI, often resulting in a drop in transduction coverage compared to just one vector.   Q2: ...between ddPCR and qPCR? A: At CLOVER, we have traditionally used qPCR (protocol link). We have found this... Q4: Are there any AAVs that have successful transduction in microglia? I have been using the AAV.PHP....

...through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded overhangs...higher DNA concentrations are needed for this method to be successful. Low DNA concentration? No problem...overlapping regions of single-stranded DNA. But as long as the double-stranded region is long enough to hold...LIC) was first developed in the 1990s. While traditional restriction enzyme cloning uses short sticky ...nicks easily repairable by the bacteria after transformation. LIC is a reliable cloning method, but it is... is limited by its sequence constraints. The long overhangs must not contain the dNTP present in the exonuclease...ligation-independent cloning (SLIC), eliminated many of LIC’s constraints by relying on the power of homologous recombination...

...seven-(pass)-transmembrane domain receptors, due to the seven alpha helices that transverse the cellular...induce or inhibit intracellular signaling cascades as a response to the extracellular stimuli. Ligand dissociation...fusion to a receptor-transcription factor fusion. TEV protease cleave the transcription factor from the receptor...initiating signaling pathways in inflammation, and neurotransmission. These receptors can be activated by an array...of different ligands, including hormones, neurotransmitters, photons or odorants, to elicit downstream...coupled with heterotrimeric G proteins on the intracellular side of the cell membrane. Heterotrimeric G ...proteins are important molecular switches in signal transduction pathways and consist of a trimer of three subunits...

...to a great image is contrast. The human visual system relies primarily on contrast to identify individual...around us. Without contrast, objects simply vanish into noise. Due to its unrivaled contrast, fluorescence...fluorophores can add a second layer of contrast, color contrast, to an image and thereby provide molecular...deeply penetrating infra-red excitation light can push imaging depths closer to 1 mm. Traditionally, 2-photon... is not possible via traditional confocal due to limitations in light penetration mentioned above (5)....imaging has emerged as the dominant light microscopy contrast technique in modern biology (1). When performed...correctly, fluorescence microscopy provides a high contrast image in which a bright signal overlays a perfectly...

...activity and instead become trapped in a covalent bond with its substrate. Then, all it takes is a bit...range of useful molecules linked to that substrate. If the substrate was linked to, say, a chemical dye or...applications.   Figure 1: Live-cell labeling strategies. The green structure represents a fluorophore...a look at some popular self-labeling tools: Tetracysteine: The first reported chemical labeling tag, from... gets covalently linked to derivatives of its substrate O6-benzylguanine (BG) (Keppler et al., 2003). ...The Virginia Cornish and Michael Sheetz labs demonstrated in 2005 that proteins fused with E. coli dihydrofolate...be labeled with fluorescent derivatives of its substrate trimpethoprim (TMP-tag) (Miller et al., 2005)....

...and GFP reporters, as well as multiple transient and transgenic systems, Sahoo et al. showed that gene...commonly used 35S promoter. Transgenes are also efficiently inherited in both transgenic tobacco and Arabidopsis...tumor-inducing” or Ti plasmid, which allows it to transfer genetic material into the host plant genome. Scientists...Scientists have worked to optimize this system for gene transfer, studying the stability of modified Ti plasmids...a new and versatile binary Ti vector for both transient and stable gene expression applications in plants...borders specify the region of DNA that will be transferred to the plant, similar to viral vectors’ terminal...binary vectors are quite large (>10 kb.) Since transformation efficiency is inversely proportional to size...

...changing its double-strand cut to a single-stranded nick. In order to produce a double stranded break, two sgRNAs...place on the free strand of DNA and converts a C to U without producing a double strand break.   Prime... target DNA. The reverse transcriptase polymerizes DNA onto the target strand based on the sequence of... using dCas9 in bacteria demonstrated that targeting dCas9 to transcriptional start sites was sufficient...approach is that two sgRNAs targeting opposite strands must be available in moderate proximity to each...desired edit adjoined to the sgRNA, a reverse transcriptase, and a nCas9. While this system has been shown...been exploited to localize factors such as transcriptional activators and repressors to target genes. ...

...lentiviral vector to be transported retrograde and also has higher neuronal transduction efficiency and a better...Callaway EM (2007) Monosynaptic Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons...Lentiviral production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the...the packaging plasmid. These are transfected into target cells which produce lentiviral particles.   ...preparation, VSV-G is stable and can withstand ultracentrifugation which helps scientists achieve a high titer... titer is one of the reasons VSV-G is used to transduce stem cells and neurons. However, VSV-G’s broad...for off-target infectivity as it can bind and transduce many cell types before reaching the target cells...

...can readily fold into transcriptionally active double-stranded form through intra-molecular annealing. ... Figure 2: DNA rescue and transduction of a conventional single-stranded AAV (ssAAV) and a self-complementary...genome conversion in the transduced cell nucleus, the single-to-double stranded conversion of the DNA goes...inter-molecular annealing or second strand synthesis. In contrast, a scAAV vector with half the size of...Adeno-associated virus (AAV) is a single stranded, Parvoviridae DNA virus, packaged in a non-enveloped...and in vivo laboratory experiments ranging from transient gene expression in a particular cell type to CRISPR...hijack host cell machinery, and synthesize a second strand of DNA before expression of their cargo can begin...

...22567149. Pubmed Central PMCID: PMC3342243. 4. Mathur, Ambika, et al. "Transforming training to reflect the...workforce." Science Translational Medicine (2015): 285ED4. Pubmed PMID: 25925677. Pubmed Central PMCID: PMC4484856...information on the symposium as a whole and for registration please go to http://futureofresearch.org/boston.... The Future of Research Symposium A year ago, trainees from around Boston met to discuss their visions...workforce is pyramidal/feudal generating too many trainees per PI” “Lack of awareness about how the system..., less than 15% of postdocs had achieved tenure-track positions within 5-7 years of earning their PhDs...first round of Broadening Experience in Scientific Training (BEST) awards, which are designed to support new...

...affiliated TniQ protein recruits transposition cofactors. The transposon proteins integrate a payload sequence...Evolving CASTs for new contexts Natural transposons evolve under contrary selection pressures: high efficiency... like we finish writing about one dual CRISPR-transposon system before another exciting new advance emerges...combined with the large sequence capacity of transposons open whole new worlds to explore. The newest ...called evoCAST — an evolved CRISPR-associated transposase (CAST). Back up a second. What are CASTs? CASTs...bacteria. In the course of evolution, Tn7-like transposons captured and repurposed nuclease-deficient CRISPR-Cas...Conveniently, the process doesn’t cause double-stranded breaks and can deliver large sequences and even...

...DA and other neurotransmitters in the brain has been in vivo microdialysis of extracellular fluid (Darvesh...PMID: 4403305. PubMed Central PMCID: PMC426888. Patriarchi, Tommaso, et al. "Ultrafast neuronal imaging of...dopamine (DA), a well-known reward chemical and neurotransmitter. But aside from DA’s famous role as the “happy... across the semipermeable membrane from the extracellular fluid into the perfused liquid. The probe then...is not suited to detect rapid changes in DA concentration. To address this, researchers from some labs...responses (increases in fluorescence) at low concentrations (e.g., 100 nm) of DA. In addition, they exhibit...following types of data: Report time-course and concentration of released DA in mouse brain slices Visualization...

...lateral”) gene transfer. Horizontal gene transfer can occur naturally through transformation (cellular uptake...? Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection Resources on Addgene.org...environment), transduction (viral infection by bacteriophages), or conjugation (direct transfer from one cell...also promote gene transfer in soil bacteria! (Demanèche et al., 2001) Beneficial traits of natural plasmids...passengers. Most natural plasmids encode beneficial traits to entice their hosts to keep them around and even...one popular method scientists use to promote transformation, but it isn’t just for lab specimens — there...humans only noticed last century! Nevertheless, the transfer of plasmids carrying these genes between bacteria...

...PubMed Central PMCID: PMC314833. Datta, Naomi. “Transmissible Drug Resistance in an Epidemic Strain of Salmonella...factors that could be transferred between cells. At the time, these extranuclear agents of heredity were...and the labels applied to them were vague or contradictory, owing in part to the fact that very little ...Latin for 'it'), as “a generic term for any extrachromsomal hereditary determinant”. His proposal, however...1960s when scientists began to study other extrachromosomal particles, particularly Resistance or R-factors...R-factors. Like F-factors, R-factors could be transferred between bacteria via cell-to-cell contact; however...experiment. Dr. Cohen and colleagues treated a tetracycline resistant plasmid, pSC101, and a newly developed...

...only have to worry about cloning your transfer plasmid. The transfer plasmids have different packaging capacities... any HEK-293T cells. If you titrate your viral preps, lentiviral titration requires an additional couple...of days, as it requires transduction of more cells, while AAV can be titrated following purification using...the virus, or go straight into your viral application, like protein production. Titration of baculovirus...Purification Ultracentrifugation (AAV); centrifugation (lenti) Centrifugation Titration time ~1 day ...AAV and lentivirus requires three plasmids — a transfer plasmid containing your gene of interest plus ...in mammalian cells is relatively quick. After transfection, HEK-293T cells start producing virus within...