...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...

...from yeast (1). It is a Drosophila geneticist’s main workhorse to turn genes on or off. Gal4 is a transcriptional.... B. Duffy, GAL4 System in Drosophila : A Fly Geneticist’s Swiss Army Knife, Genesis 34, 1–15 (2002).PubMed...H. Brand, The GAL4 System : A Versatile Toolkit for Gene Expression in Drosophila, 3, 1–10 (2015). PubMed...he still reads “#” as a “pound symbol”. References 1. A. H. Brand, N. Perrimon, Targeted gene expression...-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 ...

...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... lactose analogue, IPTG (isopropyl β-D-1-thiogalactopyranoside). (To be completely accurate, IPTG binds... 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...

...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...screening blog post for more information.   Figure 1: Result of a blue-white screen. Image from Stefan ...Because Sanger sequencing can usually sequence just 1 kb of DNA, custom primers are especially helpful in...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...

...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...fluorophores are constantly being engineered. Table 1 below lists a few common fluorescent proteins and ...definitions found in Table 2 may not apply. Table 1: The specific mutations comprising common fluorophores...

...  Plasmids for cGAL and split cGAL systems The Sternberg lab in 2016 created cGAL, a GAL-4 based bipartite... MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust GAL4–UAS system for Caenorhabditis...this system a promoter controls the expression of a GAL-4 driver that can bind to activation sequences upstream...expanded on this tool by splitting cGAL4 in two and binding each half to a gp41-1-N-intein. Split inteins are...Zhang et al., 2015). In this system a modified TIR-1 protein can degrade degron-tagged proteins in an auxin...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...biological questions. The two most common uses are: 1) to test the expression level in a specific system...brightness is compared to that of EGFP which is set as 1. Some proteins are very dim (e.g. TagRFP657, which...Photostability can be as short as 100ms (EBFP) or as long as 1 hour (mAmetrine1.2). However, for most FPs it is a...

...' mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1 gyrA96 gal-thi-1 supE44 λ-relA1 Δ(lac-proAB)/F' proAB+lacIqZΔM15...Table 1 below outlines a few of the more common genetic changes found in E. coli strains. Table 1: Common...resistance gal Mutation in galactose metabolism pathway Cells cannot grow on galactose only gyrA, ...(rB- mB-) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ- JM109   General cloning...mcrB mrr hsdS20(rB-, mB-) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 leu mtl-1 Stbl4   Electrocompetent cells...amber (UAG) stop codon by tyrosine insertion λ-thi-1 or thi1 Mutation in thiamine metabolism Requires exogenous... all based on E. coli K-12 and are considered BSL-1. Table 2: Lab strains of E. coli Strain Natural...

... assembly reaction** 1 5 min., 37 ºC (2.5 μl) 687 100% 274,200 2,742,000 1 60 min., 37 ºC (2.5 μl)... a blue colony phenotype upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction...expected complete sequence for the lacI/lacZ genes (1), while sequencing of white colonies showed a mixture...representative transformation plates obtained from 1-, 12- and 24-fragment assemblies of the lacI/lacZ ...lacZ cassette, and illustrates how the volume of the 1 ml outgrowth spread on each transformation plate can...fidelity studies conducted by Potapov et al. at NEB (1, 2). While T4 DNA Ligase, the mainstay of most biotechnological...over 90% for 24-fragment assemblies (Figure 3, Table 1). These improvements (5- to 12-fold increases in the...

...HIV-1 were transduced with a lentiviral vector containing an antisense sequence against the HIV-1 envelope... et al. transduced rat neurons in vivo with beta-gal and other reporters, and they showed that expression...Trono and collaborators turned to the lentivirus HIV-1, well-known to infect non-dividing cells. The first...plasmids. The packaging plasmid carried a mutated HIV-1 provirus that couldn’t package itself due to a few...plasmid encoded the desired transgene flanked by HIV-1 long terminal repeats (LTRs) that facilitate viral... targeting and tracing cells in the brain (Figure 1). 2nd generation lentiviral packaging system The graphic...infectivity, is often used as a substitute for HIV-1 Env, which can only infect CD4+ cells. 3rd generation...

...attract fish for them to prey upon (Figure 1). Figure 1: The sea anemone's red color can be used ...techniques such as  blue-white screening require X-gal, an expensive exogenously-added substrate, chromoproteins...synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036-018-0100-0 Murakoshi...expression in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-48604-4 Salih...

...and GAL 80. TEF1 General expression mRNA Yeast transcription elongation factor promoter Constitutive  ...mammalian expression from human elongation factor 1 alpha Constitutive  Tends to give consistent expression...Drosophila promoter conaining Gal4 binding sites Specific Requires the presence of Gal4 gene to activate promoter...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...

...disadvantages: Table 1 (Joglekar et al., 2017)  Table 1 (Gutierrez-Guerrero et al., 2020) Table 1 (Cronin et al...polyA tail.   Figure 1: Lentiviral production uses three plasmids: (1) The transfer plasmid, (2)... specific purposes as described previously (Table 1, Gutierrez-Guerrero, et al, 2020). Because retrovirus...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 ...

...intracellular targets. Nature Communications, 7(1), Article 1. https://doi.org/10.1038/ncomms10778 Erdmann...molecules in vivo. Nature Biotechnology, 21(1), Article 1. https://doi.org/10.1038/nbt765 Lavis, L. (2016... envelope design. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-40788-8 ... capping protein. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-39655-3 ...up linked to whatever molecule you choose (Figure 1). Just express the fusion protein and add a specific...proteins, opening up new applications.   Figure 1: Live-cell labeling strategies. The green structure...minimally-disruptive genetic tag on the protein of interest. Table 1: Summary of popular self-labeling tag systems ...

...Bacillus. Appl Microbiol Biotechnol. 2019 Jan;103(1):303-313. doi: 10.1007/s00253-018-9485-4. Epub 2018...and clonal analysis in Drosophila.” Nat Methods 9(1) (2011): 47-55. PubMed PMID: 22205518. PubMed Central...Methods Mol Biol. 2016;1478:53-78. doi: 10.1007/978-1-4939-6371-3_3. PMID: 27730575; PMCID: PMC5270762. ... and lac promoters.” Proc Natl Acad Sci U S A. 80(1) (1983): 21-5. PubMed PMID: 6337371. PubMed Central... 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- ...

...authentic research experiences to undergraduate students (1, 2), but educators face significant challenges in ...6). Methionine synthesis in S. cerevisiae (Figure 1) occurs through a well-characterized pathway with ...sequences for ten different MET/Met genes (Figure 1) from the two yeast species into the pYES2.1/V5-His...selected from different parts of the Met pathway (Fig. 1). In addition to the complementation experiments that...about Clare at the Biology Dept. site.  References 1. American Association for the Advancement of Science...genes from the pYES2.1 plasmid is controlled by the GAL1 promoter. The cloning procedure adds both V5 and... to grow in the absence of methionine. Since the GAL1 promoter controls MET/Met gene expression, complementation...

...enzyme (among others): caveolin 1, also known as CAV 1. Usually, caveolin 1 promotes the cell life cycle ...caveolin 1, there’s less malignancy because there’s less proliferation. However, the lack of caveolin 1 in ...believes that miRNA may decrease the expression of TAK 1, an enzyme that controls apoptosis and regulates transcription...Agnieszka K., et al. "An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor...museum "Fábrica Centro Ciência Viva" in Aveiro, Portugal. If cancer was a musician, then metastasize is...museum "Fábrica Centro Ciência Viva" in Aveiro, Portugal. She is particularly interested in science journalism...

...were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic, because the DREADD...plasma membranes in both species (bottom panel in Fig. 1). Our results indicate that the tag protein may have...protein of interest the membrane.   Figure 1: Electron microscope images showing ultrastructural...This post was contributed by Adriana Galvan, an associate professor at Emory University School of Medicine...express hM4Di in neurons of rhesus monkeys or mice (Galvan et al., 2019). The hM4Di receptor is an engineered....     Thank you to our guest blogger! Adriana Galvan is an associate professor at Emory University School...2159–2165 . https://doi.org/10.1128/mcb.8.5.2159 Galvan A, Raper J, Hu X, Paré J, Bonaventura J, Richie...

...for the wild-type. However, variants such as the P.1 (Gamma variant) and the B.1.351 (Beta variant) were... tested at the time of this study. Figure 1: Neutralization levels against spike pseudovirus decreases...lowest levels of cross-neutralization occurs in the P.1 and B.1.351 variants tested. Image from Garcia-Beltran...References Cele S, Gazy I, Jackson L, Hwa S-H, Tegally H, Lustig G, Giandhari J, Pillay S, Wilkinson E...Hanekom W, Moosa M-YS, Lessells RJ, de Oliveira T, Sigal A (2021) Escape of SARS-CoV-2 501Y.V2 from neutralization...Feldman J, Pavlovic MN, Gregory DJ, Poznansky MC, Sigal A, Schmidt AG, Iafrate AJ, Naranbhai V, Balazs AB...

...Weight Markers. Scientific Reports,7(1). doi:10.1038/s41598-017-02693-1. PubMed PMID: 28550309. PubMed Central...plasmids that can be used to make inexpensive 100 bp or 1 kb DNA molecular weight ladders were recently deposited... PstI or EcoRV, these plasmids generate 100 bp or 1 kb DNA ladders, respectively. Unlike many commercially... gels. Get Tips on Verifying Your Plasmid Table 1: Comparing Typical Comericial Ladders with the PSU...$10 to make 1000 lanes worth of the 100 bp or the 1 kb ladder. Using a commercial plasmid prep kit increases...Learn How to Analyze a Restriction Digest References 1. Henrici, R. C., Pecen, T. J., Johnston, J. L., & ...Additional Resources on the Addgene Blog Are you a Frugal Scientist? Save Money with the Baby Gel Box Find...