...Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm...below affect continuous cellular processes (e.g., protein synthesis), while amp only affects the cell wall...Kanamycin, or kan, is an aminoglycoside which inhibits protein synthesis. The kan resistance gene, NPTII, facilitates...streptomycin Spectinomycin and streptomycin both inhibit protein synthesis as their mode of action and have a very...gene provides zeocin resistance by expressing a protein that directly binds to zeocin, preventing it from.... M., Piccinini, T. E., Lammel, C. J., Hadley, W. K., & Brooks, G. F. (1986). Effect of storage temperature...entirely detoxified, even when Sh ble is expressed (Trastoy et al). Thus, it may not be a good choice for experiments...

...to modified proteins called histones. We can detect modified histones with “GPS for proteins,” ChIP-seq... compute coordinates for genetic elements like protein, RNA, and DNA (Table 1). As with GPS in the real...techniques Technique What It Locates ChIP-seq Proteins (for our example later, histones) ChiRP-seq ...For details on DNA compaction and the structural proteins that make it possible, see the appendix DNA is... you run a ChIP-seq experiment (think “GPS for proteins”) for NANOG, an essential transcription factor...epigenomics: H3 – one of NANOG’s associated histone proteins K4 and K27 – locations of the amino acid lysine... is a combination of coiled DNA and structural proteins called histones. Chromatin folds into secondary...

...elegans. Other groups have used Mango to study RNA-protein complexes (Panchapakesan et al., 2017) and short...selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution...highly efficient RNA mimics of green fluorescent protein." Nature structural & molecular biology 21.8 (2014... Resources at Addgene.org Browse fluorescent protein plasmids and resources See all Jaffrey lab plasmids...additions to aptamer soup! References Alam, Khalid K., et al. "A fluorescent split aptamer for visualizing...developed to quantify RNA transcription by RNA Polymerase III. By expressing Corn under promoters for transcripts... transcripts uniquely transcribed by RNA Polymerase III (5S, tRNA and U6) and measuring Corn fluorescence...

...models for immunogenicity prediction of therapeutic proteins. European journal of pharmaceutics and biopharmaceutics...https://doi.org/10.3389/fddsv.2022.952326Joubert, M. K., Deshpande, M., Yang, J., Reynolds, H., Bryson, C...This allows scientists to get a more definitive grasp on the immunogenicity of the drug product while ...A Method Using Existing in silico Algorithm Infrastructure and a Vision for Future Enhancements. Frontiers...

...harmful weeds. The parasitic fusion is such that the two plants share RNA, proteins, and DNA. Euprymna...manipulated by injected RNA and can express fluorescent proteins. This lab has created an automated tank for feeding...the brain allows the squid to create a different protein. Did you know that most cephalopods die after they...systems for scientists and educational programs for K-12 students. You can see why I was excited to be invited... "for development of tools, approaches, and infrastructure necessary for direct tests of cause and effect...) -  perhaps you’ll choose to study Crepidula atrasolea or Prochlorococcus with your next project. Gymnotiformes...protocol community! Asclepias syriaca and Asclepias curassavica (Gerog Jander) Milkweed plants are known to many...

.... Once a co-repressor protein binds the repressor protein, the repressor protein can bind to the operator...activator protein is bound, turning transcription ON. When a repressor binds the activator protein, the activator... and largely defines the amount of recombinant protein that will be produced. Many commonly-used promoters... activator protein cannot bind the promoter sequence anymore and transcription is turned OFF. Negative...Negative Repressible: A repressor protein is present, but cannot bind the promoter sequence and transcription....1038/s41598-022-07852-7 De Boer, Herman A., Lisa K. Comstock & Mark Vasser. “The tac promoter: a functional...Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region ...

...at the Institute for Protein Innovation (IPI), a Boston-based, nonprofit protein research institute, partnered...Table 1: IPI antibodies  Antibody Target Proteins Reactivity Isotype Source Species Anti-Integrin...plasmids. To encourage collaboration and education in protein science, IPI hosts events and training courses ...Faulds is a science writer at the Institute for Protein Innovation. She writes about all things IPI, from...References Hao, Y., Yan, J., Fraser, C., Jiang, A., Anuganti, M., Zhang, R., Lloyd, K., Jardine, J., Coppola...and morphed asymmetrically (Jo et al., 2022). By contrast, LVDP-binding integrin α4β1 required and conferred...

...allows you to ask questions like: “Where does my protein of interest live within a cell,” “Does this disease...where they bind to their targets, most commonly proteins. You can then use fluorescent microscopy to look... multiplexing is that you can ask how multiple proteins relate to one another in the same sample. For ...dozens of products that all claim to target your protein of interest. Thankfully, there are a number of ...experiment.    References and resources References Im K, Mareninov S, Diaz MFP, Yong WH (2018) An Introduction...allowing one to “directly” visualize their target. In contrast, indirect IF requires two antibodies: a primary...

...bacterial strain that is deficient in recombinase protein RecA can reduce the frequency of multimerization...large inserts are often dimers in Escherichia coli K-12. Plasmid. 1989 Mar;21(2):138-41. doi: 10.1016/0147...microbiolspec.PLAS-0025-2014. PMID: 26104344. Fishel RA, James AA, Kolodner R. recA-independent general genetic...

...approaches to study protein interactions is the pull-down assay, wherein a protein of interest can be ...IP/MS, a target protein is first immunoprecipitated - along with its associated protein complexes - from...using antibodies against the target protein. The captured protein complexes are then analysed by mass ...interacting proteins. Similarly, in ChIP-seq assays, chromatin fragments that are bound by a protein of interest...target protein. Both IP/MS and ChIP-seq methods provide a wealth of information about how protein networks...pre-validated antibody for each target protein. To study 20,000 human proteins, this would be time-consuming, ...tag the protein of interest with a small biochemical “epitope tag” such that these tagged proteins can be...

...reporter constructs), in functional analysis of proteins (to perform alanine scanning mutagenesis or targeted...sites. Alternatively, if you want to make your protein non-functional, you may simply introduce a frame-shift...PMID: 8470801. PubMed PMID: 8470801. 3. Saiki, R. K., et al. "Primer-directed enzymatic amplification ...you need a high-fidelity polymerase. There are many high fidelty polymerases on the market; you need one...a DNA polymerase that produces blunt-ended PCR products (e.g. Phusion, Pfu, and Vent polymerases). Note...amplified fairly easily by simply following the polymerase manufacturers’ PCR protocol. Be sure to adjust...plasmid template for site directed mutagenesis.  Polymerase To ensure that you don’t introduce undesired ...

...give rise to an adenine base editor. A massive protein engineering effort by Gaudelli et al. resulted ... restore antibiotic resistance. To find such a protein, they created a mutant library of E. coli tRNA ... ABE does not require any additional inhibitor protein like UGI in CBE. Base editors for the plant genome...probably due to the presence of nucleosome or other proteins. You can detect base editing as early as 48-72...29807545. PubMed Central PMCID: PMC5972399. 12. Molla, K., Shih, J. & Yang, Y. Multiplex CRISPR-mediated base...inosine), which is treated as ‘G’ by cellular polymerases. Subsequently, an original genomic A-T base pair...

...other small base pair changes, and the addition of protein tags. It also has the flexibility to modify the...: 1) use a strain of bacteria that has key MMR proteins knocked out or 2) specially design ssDNA oligos... not the amino acid sequence of the translated protein.  These changes can be 5’ or 3’ of the desired ... detail below).  Controlling expression of Red proteins is critical for minimizing the toxic effects of...10811905.PubMed Central PMCID: PMC18544. 3. Murphy, K. 2016. λ Recombination and Recombineering, EcoSal ...105 colonies. Pro Tip: Use a high-fidelity Taq polymerase with proof-reading ability for generating dsDNA...nucleotides 1 out of 104 to 105 Use a high fidelity Taq polymerase to generate insert ssDNA Point mutations or...

...repair proteins fused to CGBEs The David Liu lab also experimented with adding different proteins to the... binding motif protein X-linked (RBMX) (Koblan et al., 2021). Several of these proteins had additive effects... the DNA repair protein rXRCC1, or the DNA binding and lyase domain of DNA polymerase β (PB) (Chen et ...UdgX, they found three proteins whose fusion improved the CGBE: DNA polymerase D2 (POLD2), exonuclease... cytosine transversion editors. New DNA repair proteins fused to CGBEs Instead of using UNG, the Wei Leong...improving C → G editing. Instead of fusing DNA repair proteins to the base editor, they added the pioneer factor...with CRISPR-Cas9-directed base excision repair proteins. Nature Communications, 12(1), 1384. https://doi.org...

...marks for safety.   Protein Delivery: Delivering reprogramming factors as protein is another way to avoid... vector-free Multiple transfections required Protein Delivery DNA-Free Low High Transgene-free and vector-free... inefficient Difficult to purify reprogramming proteins. Table 1: Key Features of Different Methods...are safer than integrating methods, with RNA and protein delivery considered the safest since there’s minimal... exposing cells to exogenous genetic material. Proteins are delivered to cells by fusing them with peptides...Kim et al, cells were exposed to reprogramming proteins for 8 hours a week for 6 weeks, and still required...and it’s difficult to purify the reprogramming proteins needed to perform these experiments. Assays for...

...in a direct or unbiased way at the DNA, RNA, or protein level. The “correct” choice of tool requires careful...unmatched—unlike CRISPR experiments that require exogenous protein, successful induction of RNAi only requires the...scored. However, determining the transcript or protein abundance of your specific target required physical...Bush J, McCormack M, Sheen J (2013) Comprehensive Protein-Based Artificial MicroRNA Screens for Effective... M, Grandi P, Jakob P, Michon A-M, Sun H, Tessmer K, Bürckstümmer T, Bantscheff M, Steinmetz LM, Drewes... a targeting sequence for the adenine phosphoribosyltransferase (APT) gene, which is conserved in many...

...the same page in terms of the vocabulary--DNA, protein, plasmid, gene, etc. Though I talked about CRISPR...teach  5-10 year olds in a single evening. Grades K-5 cover a broad range of attention spans, skill levels... I came up with an analogy that would help them grasp some of the key concepts (Fig. 2). One thing I will...

... be used as a proxy measure for differences in protein expression. (If you're just getting started, check... amplicons. Image credits (respectively): Zuzanna K. Filutowska; Selma, 2019; Trampuz, 2003.   You...doi.org/10.1038/s41598-017-02217-xTaylor, SC; Nadeau, K; Abbasi, M; Lachance, C; Nguyen, M; Fenrich, J. The...References and Resources  References Biassoni R; Raso A. (eds.), Quantitative Real-Time PCR: Methods and...

...components including (1) a CRISPR RNA, (2) four proteins forming the QCascade DNA-targeting module with...with the crRNA, (3) three transposase proteins, and (4) the donor DNA. By using a multi-spacer CRISPR array...Zalatan’s lab used an approach to identify known proteins involved in the activation of transcription including...including transcription factors, phage proteins, and RNAP components (Dong et al., 2018). They found a...dCas9 variant fused to bacterial enhancer binding protein required for the activation of sigma-54 type promoters...glycosylase inhibitor and degradation tag on the fusion protein to limit the activity of the system as a whole.... Rep 6: . https://doi.org/10.1038/srep19452 Selle K, Fletcher JR, Tuson H, Schmitt DS, McMillan L, Vridhambal...

...complexes to study neural function. Often the reporter proteins associated with these complexes are so robustly...vectors: packaging capacity and the role of rep proteins in vector purity." Human gene therapy 18.2 (2007... 1248. PubMed PMID: 11509958. 8. Ferrari, Forrest K., et al. "Second-strand synthesis is a rate-limiting...Surprisingly, little research has compared and contrasted optogenetic and chemogenetic receptors delivered...

..., Manning CF, Nguyen JT, Bechtold H, Dumitras C, Gong B, Nguyen K, van der List D, Murray KD, Engebrecht...exciting for scientists working to visualize multiprotein complexes.  Fig. 2. Multiplex immunolabeling...

...cancer.     References 1. Russell, David W., and Roll K. Hirata. "Human gene targeting by viral vectors." ...devised methods to deliver CRISPR components as ribonucleoproteins (RNPs) that can be supplied separately from.... PubMed Central PMCID: PMC4752446. 5. Asuri, Prashanth, et al. "Directed evolution of adeno-associated...28646206. PubMed Central PMCID: PMC5482879. 7. Bak, Rasmus O., and Matthew H. Porteus. "CRISPR-mediated integration...