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...MTRIA for short), where a fluorescent protein is inserted into one of the loop regions of any G-protein-... Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted GFP into an intracellular...calcium reporter Near-infrared (NIR) fluorescent proteins promise better performance for in vivo use and...indicator using small monomeric NIR fluorescent proteins (miRFP670nano and miRFP720) and the Ca2+-sensing...structure-guided engineering of potassium binding proteins, they developed a family of fluorescent potassium...et al. 2022). By fusing two bright fluorescent proteins with different thermal quenching rates (mNeonGreen...Additional resources on the Addgene blog Fluorescent Proteins 101: Fluorescent Biosensors Resources on Addgene.org...

...where they bind to their targets, most commonly proteins. You can then use fluorescent microscopy to look...visualize their target. These antibodies are generated against a specific antigen and then conjugated with a fluorescent...but is not attached to a fluorescent molecule, instead a secondary antibody that targets the primary is...for each primary antibody could get out of hand. Instead, it is more efficient to generate a handful of ... multiplexing is that you can ask how multiple proteins relate to one another in the same sample. For ...see Im, et al. 2019.  Fixation Good fixation maintains sample morphology with minimal impact to the target...spectral properties match your available imaging instruments and the spectral properties do not overlap with...

...reaction that cleaves a nearby RNA sequence that contains a fluorophore that is quenched when intact and...improves sensitivity of the assay. This reaction also contains SSB, strand displacement DNA polymerase, recombinase...fluorescence visualization. In this technique, however, instead of using lateral flow strips to detect the cleavage...Reverse-transcribed RNA samples are amplified by PCR instead of RPA. Unlike RPA, this technique uses Taq polymerase... which is widely available and less expensive. Instead of using the typical PCR machines, the authors ...depend on collateral cleavage by the enzyme and instead relies on its activity on the target regions thus...COVID-19 using CRISPR diagnostics. https://www.broadinstitute.org/files/publications/special/COVID-19%20detection...

... directed evolution to get them to work on DNA instead. However, they retained a small amount of RNA-editing... mutation. To tackle this problem, Benjamin Kleinstiver’s lab engineered new variants of S. pyogenes Cas9...came out on top. Narrowing PAM preferences The Kleinstiver lab also worked on the opposite project: engineering...tested mutations produced efficient editors, the Kleinstiver set performed best when paired with the V106W...Silverstein, R. A., Hille, L. T., Ogul, A. N., Robinson-Garvin, N. A., Small, J. C., McCague, S., Burke...Urbina, E. O., Dahl, G. A., Correia, B. E., … Kleinstiver, B. P. (2025). Custom CRISPR-Cas9 PAM variants... R. T., Christie, K. A., Whittaker, M. N., & Kleinstiver, B. P. (2020). Unconstrained genome targeting...

...plasmid, into which the GOI is inserted, and a Ti "helper" plasmid, which contains Vir genes necessary for T-DNA...gRNA scaffold sequence and allow researchers to insert targeting oligos with minimal cloning required....Dicot Genome Editing Kit from the Stuttmann lab contains a variety of Agrobacterium-compatible, Cas9 containing... T-DNA processing and insertion into the plant genome. B) The Ti plasmid and helper are transformed into...the Vir genes on the helper plasmid and used to insert the intervening region into the plant genome. Created...- promoters used, precise protein sequences or domains, and methods of delivery - the underlying technique...

...studies or make a reporter construct. Fluorescent proteins (FPs) are also the foundation of multiple important...biological research. Photoconvertible fluorescent proteins Newly developed photoconvertible FPs like PsMOrange...repeat the process with these new photoconvertible proteins. With Sattarzadeh et al.’s discovery that GFP ...After exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. noticed that GFP ...encoding one of the unexpectedly photoconvertible GFP proteins to make a new transgenic line could avoid problems...as well as a wide variety of other fluorescent proteins for your imaging needs. If you need an FP refresher...

...Llamas Required - Synthetic Nanobodies Against Membrane Proteins More resources from addgene.org  Plasmids...single-domain antibodies genetically fused to fluorescent proteins and used to visualize target antigens in living... transgenic mice that express camelid variable domains (aka nanomice), which gives researchers the “best...made to generate the nanomouse. (VHH = variable domains of heavy chain only antibodies) (B) Nanomice express...widely adopted. So thank you to the camelids who inspired this versatile family of tools, the applications...

...and to disrupt endogenous genes at the site of insertion. In both of these cases, transposons combine the...blue) in a plasmid backbone. A separate plasmid contains the transposase gene (red) for expressing the ...sequence is duplicated near the gene of interest insertion site.   The Sleeping Beauty transposon system ...within the genome. The Sleeping Beauty transposase gains its name from the fact that it was recreated from...continues to be altered in order to improve its gene insertion efficiency in primary cells. In 2009, Zsuzsanna...or 5’ end of the gene of interest allowing for insertion in the correct orientation. These plasmids use...

...Carolina (plasmids at Addgene). It consists of two domains, LOV2 and Zdk1, where Zdk1 binds LOV2 with high...normally sits on microtubules to attract other proteins involved in their growth and extension. Now, they...domain. Each half was fused with one of the LOVTRAP domains. When both halves of the modified EB1 protein are...inactivated by the separation of its synergistic domains. This paper demonstrated that the migration of ...fluorophore binding aptamers and MS2-binding fluorescent proteins were the state of the art in RNA imaging in vivo...on the lookout for better, brighter fluorescent proteins (FP) with improved tissue penetrance and live ...

...Argonaute proteins are also present in prokaryotes, and some of these proteins protect against invading...reasons why you might choose NgAgo over CRISPR proteins Cas9 or Cpf1 - keep reading to learn more! Background...: Argonaute and NgAgo In eukaryotes, Argonaute proteins play an important role in RNA interference, binding...when the protein is being synthesized, so NgAgo remains faithful to its guide DNA and cannot switch guides...

...usage has been limited by technical problems. For instance, it’s much more difficult to introduce a gene ...strategy for transducing primary cells. What if, instead of supplying a DNA construct, they could simply... adapted from D’Astolfo et al. In certain instances, the potential benefits of nonviral transduction...with virus work. Fourth, working directly with proteins removes the limitations of packaging size associated...References Efficient Intracellular Delivery of Native Proteins. D'Astolfo DS, Pagliero RJ, Pras A, Karthaus WR...PubMed. Find the Plasmids @Addgene: iTOP-tested proteins and reporters Previous Protein Delivery Techniques...

...Tom, and Alan E. Tomkinson. "Eukaryotic DNA ligases: structural and functional insights." Annu. Rev. Biochem...many molecular biology protocols, including vector-insert joining for recombinant plasmid construction (restriction...DNA ligase, are naturally able to discriminate against ligating substrates containing base pair mismatches... can be achieved through a combination of the intrinsic selectivity of the ligase active site and careful...indicated that the optimal buffer for Taq DNA Ligase contains 100 – 200 mM KCl at pH 8.5. This optimization....6 (2004): 757-764. PubMed PMID: 15582400. 2. Tomkinson, Alan E., et al. "DNA ligases: structure, reaction...

...freeze-dried, cell-free (FD-CF) components. Making proteins outside of a cell - cell free extracts Typically...transformation), essentially giving the cell all the instructions it needs to produce that green fluorescent protein...systems contain all the parts needed to produce proteins without the need to handle a living, replicating...simply need to add water to express a variety of proteins. FD-CF reactions are also cost-effective, as each...easily sense, whether that’s bright fluorescent proteins that they can see, enzymes that can convert certain...activities fit your lesson plans. If you are an instructor at the college level, perhaps consider using ...

...accumulate inside the producer cells and purification protocols often describe a process that begins with cell... carried inside the vesicle, which may also contain other cellular molecules such as proteins or nucleic...cells also contain cellular macromolecules (e.g. proteins, nucleic acids) which possibly improve transduction... contexts, such as the delivery of therapeutic proteins to the brain which requires crossing the blood-brain-barrier...

...but can also be used to brainstorm on data in smaller groups, where for instance the presence of the PI...lab meetings with a focus on discussion and brainstorming Though, we might be forced to work from home...of each talk - time for free interaction and brainstorming. This could be achieved by asking each presenter...problem! You can still creatively collaborate and brainstorm! Image from Joseph Mucira.   Implement new... be written by several authors from different institutes, disciplines, and cultural backgrounds.” This...

...confirm the presence of an insert in a particular vector by excising the insert from the backbone. To do...use enzymes with restriction sites that flank the insert. You will need to know both the approximate size...vector backbone as well as the predicted size of the insert. You can search NCBI for YGOI to find the particular...right has a total size of 7.3kb, including a 1.2 kb insert. The plasmid was digested with 2 unique enzymes... and 1.2kb (red box), matching the backbone and insert, respectively. The results on the gel correspond...

...Associate Director of the Cancer Program at the Broad Institute of Harvard and MIT. The notion of cancer precision...We've developed a new plasmid toolkit, the Broad Institute Target Accelerator Plasmid Collection, that we...variant function and cancer phenotypes. Broad Institute Target Accelerator Plasmid Collections To overcome...this first challenge, we’ve developed the Broad Institute Target Accelerator Plasmid Collections, now widely...Associate Director of the Cancer Program at the Broad Institute of Harvard and MIT and is focused on making precision...Kits at Addgene Browse Plasmids from the Broad Institute ...

...Root from the Broad Institute and Alex Chavez from the Wyss Institute were instrumental in helping us establish...School, and Jonathan Ting’s lab from the Allen Institute have supported the AAV initiative from the beginning...Janelia Research Campus have provided valuable insight into AAV production. Finally, we are blessed to...permission from relevant plasmid depositors and their institutions to distribute viral preps derived from plasmids...

...interact with the host. This is the role of Gag proteins: they contain the matrix, capsid, and nucleocapsid...assembly, and display. Pol, the last component, contains the reverse transcriptase and integrase components...convert the cargo from RNA to DNA and permanently insert the cargo into the host genome. Lentiviral-specific... Viral packaging and entry The viral packaging proteins for AAV are rep and cap. Rep is required for viral...discussed. LXs/Iva2 are primarily capsid assembly proteins required for packaging of the virus into particles...Resources Kulanayake, S., Tikoo, S. K. Adenovirus Core Proteins: Structure and Function. Viruses. 2021 13(3):388...

...proto-oncogenes and disrupt their expression (Robinson, 1982). To add insult to injury, retroviruses generally have...This viral DNA (also called proviral DNA) is then inserted into the host's genome and is used to make more...virus-specific elements such as the LTRs or structural proteins are not interchangeable between the two systems...Adenovirus generally has high immunogenicity, but some strains have been engineered to induce a lower inflammatory...immunogenicity low! References and Resources References Robinson HL. Retroviruses and cancer. Rev Infect Dis. 1982...