...containing T7 RNAP (DE3) F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) General protein expression BL21 (DE3...origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysS (CamR) Expression of toxic proteins...origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysE (CamR) Expression of toxic proteins...transcript half-life F- ompT lon hsdSB (rB- mB-) gal dcm rne131 (DE3) General expression; not recommended...required for expression F- ompT lon hsdSB (rB- mB-) gal dcm araB::T7RNAP-tetA General protein expression...repetative sequences.  F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) Δ(srl-recA)306::Tn10 (TetR) Expression...control of expression F- ompT lon hsdSB (rB- mB-) gal dcm lacY1 (DE3) Expression of toxic or insoluble...

...Chen. Last week, the Addgene 10-year Anniversary Gala celebrated all of the people who have helped Addgene...even posed for pictures with some Addgenies. As the gala came to an end, it was clear that the Addgenies ...journey has been a tough but memorable one. At the gala, one thing was clear: the sense of family is still...lovely play-by-play of Addgene's 10 Year Anniversary Gala. The Addgene lab often works as a very well-oiled...

...expressed, the β-galactosidase enzyme can break down a dye-linked substrate called x-gal (5-bromo-4-chloro.... On the other hand, intact β-galactosidase produces pigment from x-gal (included in the transformation...enough time for any intact β-galactosidase to be expressed and process x-gal into blue pigment (16-20 hours... should be blue, indicating that your IPTG and x-gal are working as they should be. Don't rush the process...white colonies. Take care in making your plates: X-gal is light and temperature sensitive and needs to be...chloro-3-indolyl-β-D-galacto-pyranoside) into galactose and an insoluble blue pigment (4-chloro-3-brom-indigo...gene called lacZ that encodes for the enzyme β-galactosidase. Expression of the lac operon is induced by ...

...-type specificity! The Gal4/UAS system At a more detailed level, the Gal4/UAS system is a transcription...the UAS sequence are only expressed when Gal4 is expressed. Gal4 expression can be regulated by any chosen...Controlling multiple genes with Gal4/UAS system One can also use Gal4 to drive expression of multiple ...Applications of the Gal4/UAS system There are a number of fancy ways to use the Gal4/UAS system, and you... my recent paper to see the Gal4/UAS system in action (5). I use the Gal4/UAS system to knock down gene...down. To do this, Drosophila geneticists use the Gal4/UAS system. This incredibly useful, yet simple system...geneticist’s main workhorse to turn genes on or off. Gal4 is a transcriptional activator that binds to UAS...

...researcher, when the substrate X-gal is broken down by 𝛽-galactosidase, it turns into an insoluble blue...disrupting 𝛽-galactosidase activity. These cells, presumably the cell you want, cannot break down X-gal and are...transformation on ampicillin plates containing X-gal allows you to identify cells that took up the vector...on the lacZ gene. lacZ encodes the enzyme 𝛽-galactosidase which can hydrolyse lactose. Fortunately, for... is deleted, it creates a non-functional 𝛽-galactosidase. Complementing this mutation by expressing this...example on a plasmid, creates a functional 𝛽-galactosidase. Blue-white screens rely on vectors where the...

...featuring a guest blog post from Gal Haimovich of greenfluorescentblog. Gal will share his list of 10 things...experiments. In the meantime, if you haven't checked out Gal's blog, you should! He's got great articles and insight...

...  Plasmids for cGAL and split cGAL systems The Sternberg lab in 2016 created cGAL, a GAL-4 based bipartite...this system a promoter controls the expression of a GAL-4 driver that can bind to activation sequences upstream... 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust GAL4–UAS system for Caenorhabditis elegans...2018, the lab expanded on this tool by splitting cGAL4 in two and binding each half to a gp41-1-N-intein...split system, only cells that have both halves of cGAL can drive expression of your gene of interest.  ...Central PMCID: PMC5693259.  Wang, Han, et al. "Split cGAL, an intersectional strategy using a split intein...

...This post was contributed by Gal Haimovich of greenfluorescentblog. Be honest. Do you really know how...greenfluorescentblog. Thank You to Our Guest Blogger! Gal Haimovich, PhD, is a research fellow in the lab of...

...resistance gal Mutation in galactose metabolism pathway Cells cannot grow on galactose only gyrA, .... F' mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1 gyrA96 gal-thi-1 supE44 λ-relA1 Δ(lac-proAB)/F' proAB+lacIqZΔM15...Φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG fhuA::IS2 DB3.1 Streptomycin...screening, leucine auxotroph. F- endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,...plasmid after preparation. rpsL thr leu thi lacY galK galT ara tonA tsx dam dcm glnV44 Δ(lac-proAB) e14- ...- Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 λ- e14- mcrA0 relA1 rpsL150(strR) spoT1 mcrB1...(TetR) ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14-  Φ80dlacZ∆M15 recA1 relA1 endA1 nupG rpsL...

...proteins under the control of the galactose inducible promoter (GAL). Other commonly used promoters include...

...techniques such as  blue-white screening require X-gal, an expensive exogenously-added substrate, chromoproteins...

... a blue colony phenotype upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction...reconstruction of the coding sequence for beta-galactosidase in the lacI/lacZ cassette. Additional confirmation...

... et al. transduced rat neurons in vivo with beta-gal and other reporters, and they showed that expression...cells by a lentiviral vector. Naldini L, Blömer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono ... Cartier N, Aubourg P, Fischer A, Cornetta K, Galacteros F, Beuzard Y, Gluckman E, Bushman F, Hacein-Bey-Abina...

...and GAL 80. TEF1 General expression mRNA Yeast transcription elongation factor promoter Constitutive  ...containing Gal4 binding sites, commonly used in Drosophila Specific Requires the presence of Gal4 gene to...with galactose; repressible with glucose  Can be used independently or together. Regulated by GAL4 and ...mammalian expression promoter from the human cytomegalovirus Constitutive  May contain an enhancer region...expression. Modulated by calcium and calmodulin. GAL1, 10 General expression mRNA Yeast adjacent, divergently...

... system). The GAL80 repressor can bind to GAL4, partially inhibiting the binding of GAL4 to UAS. One can...domain (DBD) to GAL4 or VP16 activation domain results in chimeric GAL80-repressible and GAL80-insensitive...expression is low but is activated by GAL4 binding to UAS. If you place the GAL4 gene downstream of a tissue- ... can therefore place GAL4 and GAL80 under different promoters and create sophisticated patterns of UAS-driven...intermediate expression from QUAS, as seen with GAL4/GAL80 and UAS. QF-mediated repression is reversible...phase of yeast culture. Repressible Binary Systems GAL4/UAS In Drosophila or development studies, you may...across development. One common binary system is the GAL4/UAS system isolated from yeast. In this system, ...

...virus in cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars were less stable than ...and spumaviruses to human serum by galactosyl(alpha1-3)galactosylation. Journal of virology 71:6174–6178...express these sugars. (Takeuchi et al., 1997). The alphaGal sugars end up in the envelope and are targets ...targets for complement-based killing by anti-alphaGal antibodies. The VSV-G envelope protein is inactivated ... such as the one from gibbon ape leukemia virus (GALV) are more stable (Sandrin et al., 2002). Studying...

...gene expression stable regardless of copy number (Segall-Shapiro et al., 2018). Designing stabilized promoters...constant. In the case of the stabilized promoters from Segall-Shapiro et al., they chose a TALE protein as the...replication, the stabilized promoters created by Segall-Shapiro et al. showed nearly no position-dependent...as cell division slows (Kittleson et al., 2011). Segall-Shapiro et al. showed that the stabilized promoters...number during the transition to stationary phase, Segall-Shapiro et al. placed a trans-acting RepA protein...genes whose proteins are needed sequentially. When Segall-Shapiro et al. placed the genes under the control...these stabilized promoters a try. References 1. Segall-Shapiro, Thomas H., Eduardo D. Sontag, and Christopher...

...provide legal advice or opinions. The contents of this post should not be relied upon for legal advice ...information presented may not reflect the most current legal developments.  Further, this post may contain technical...attorney should be contacted for advice on specific legal issues.  The permanent residence “green card” process... we recommend that you reach out to experienced legal counsel. Many thanks to our guest bloggers Lauren...Immigration is a federal law, Casseus Law provides legal services to people located in all 50 states and ...

...s the heart and soul of Addgene’s mission), but legal complexities can arise when scientists are sharing...sharing space, we heard over and over again that the legal documents were one of the biggest hurdles for scientific... of not just the physical samples, but also the legal documentation that allows you to conduct your experiments...easy Even with all those benefits, we know that legal documents can get tricky. Here’s how Addgene helps...

...Drosophila, where expression of Cre from the standard UAS/GAL4 system is toxic to proliferating cells. A Cre-estrogen... recombinase with flies heterozygous for tubulin-GAL4. For Dre, Flp, KD, R, B2 and B3, approximately 50%...50% of offspring carried both the recombinase and GAL4, indicating that the recombinases were inherited...In contrast, 0/79 offspring carried both Cre and GAL4, further emphasizing the toxicity of this enzyme...