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...tethered to bring them into close proximity (Figure 1). Formation of the complete FP results in a fluorescent...detected by fluorescence microscopy. Figure 1: How BiFC works. A split FP (e.g., yellow fluorescent... protein interactions in living cells. Nat Protoc 1:1278–1286. https://doi.org/10.1038/nprot.2006.201 ...PIPs, resulting in a fluorescent readout.   Applications for bimolecular fluorescence complementation...resulting from the assayed PPI. This high-throughput application is much more straightforward to carry out because...protein construction, cellular expression, and quantification (Kerppola, 2006, 2013). These are described... supraphysiological expression levels. BiFC quantification This is where BiFC outshines FRET — BiFC experiments...

...environments such as live cells and tissues (Figure 1). These flexible strategies give you the best of both...majority of in-cell labeling strategies have two parts: (1) a genetically encoded “tag” expressed as a fusion...Follow him on twitter @rhodamine110.   References 1. Liu, Zhe, Luke D. Lavis, and Eric Betzig. "Imaging...Subsequent advances in fluorescent proteins have replicated many of the properties once exclusive to small-molecules...phosphopantetheinyltransferase) – These enzymes catalyze the covalent attachment of a fluorophore ligand...labels for fixed-cell imaging, not for live-cell applications. Thus, these fluorophores are often bulky and...the Addgene Blog Learn About Other Protein Tags Catch Up on Your Fluorescent Protein Background Which ...

... fusions that retain their individual activities (1). Researchers also discovered the modular format of...systems could be combined effectively (3). In Figure 1 for instance, the DBD of the fusion protein shown ...ubiquitin system has been devised (8). References 1. Casadaban M.J., Martinez-Arias A., Shapira S.K., ... can often be accounted for using appropriate replicates and controls (see Tips below), and by validating...two-hybrid screening Minimizing false positives: Run replicates of the experiment to reduce the likelihood of...expression and correct folding, post-translational modification, protein degradation, access to the nucleus ...co-expression of the enzyme responsible for the modification in the assay host strain can help. In order...

...a rate of up to 1 per 500-1000 bp. In comparison, the DNA replication error rate is 1 per 109 bp. Unlike...variety of biological applications. First, GMAP-compatible backbones for lentivirus LV 1-5 and retrovirus ...Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library of approved drugs against the entire kit; ...generated 5 common 30bp overlap sequences (Sites #1-5). Each overlap site encodes a unique restriction...is multiplex automated genome engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate...of GFP (SEP) by a 9 amino-acid linker (see Figure 1). The key to the biosensor lies in pH: DsRed is relatively...inactivate SEP and leave only DsRed to fluoresce (1). With variants that can be targeted to specific cellular...

...target DNA quickly and accurately.  Figure 1: Steps in PCR reaction   This process of denaturation...extension is repeated 25-35 times to exponentially replicate the target DNA of interest. This entire reaction...have been developed for different scientific applications. These PCR methods all use the same basic PCR...the final cycles which would slow or stop PCR amplification. Other times, the PCR reaction may not be 100%...quantified by qPCR. qPCR is used in a variety of applications including gene expression profiling, studying...concentration, unlike qPCR where results can vary across replicates. ddPCR can be used to quantify DNA sequences ...droplet contains the material required for PCR amplification. Following the PCR reactions each droplet is...

...This gives each guide a score ranging between 0 and 1. The cutting frequency determination (CFD score) is...converted to a weight (which ranges between 0 and 1) so that they can be used in the rank equation above...converted to a weight (which ranges between 0 and 1) (shown in the graph below) so that it can be used...HDR. Optimal mutation in-depth One the most sophisticated features of CrispyCrunch is how it mutates guide...

...microscopic resolution for studying colloidal gold (1).  The method was based on using a thin plane (sheet...Siedentopf, and R,. Zsigmondy.  (1902) Ann. Phys.  315, 1 – 39.
 A. H. Voie, D. H. Burns, F. A. Spelman. (1993...microscopy/en_de/products/imaging-systems/lightsheet-z-1.html ...of LSFM microscopes (3,4). After this initial application, Stelzer’s group described the single-plane or...brightness with increasing depth caused by light scattering and absorption.  Finally, the photomultipliers...

...the predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging...several other constructs in plasmid form. Fig. 1: Summary of the approach and outcomes of the screen...what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated potassium...-Myc [9E10] now available! Improved voltage indicator: JEDI-2P  by: Brian O'Neill  The François St-Pierre...have successfully developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput...screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors. The resultant...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell, 18, 3408...

.... In literary terms, conflict can be defined as (1) the struggle between two opposing forces or (2) the...journey even when they know the ending.    Fig. 1: We all know that the superheroes win in the end. ...formal scientific communications, they are fantastic fodder for science communication. The trick is to ... is far from the only way to communication science. Science communicators also use the hero’s journey,...this small glimpse into the world of science communication and can use what you've learned to share your...Finding Conflict in Science  In all science communication, what you are doing is telling a (nonfiction...details often aren’t included in scientific communications, but they’re firmly part of the story to the...

... Figure 1: SPARC and SPARC2 use the GAL4-UAS system along with precisely truncated attP sites to... several common genetic tools in Drosophila (Fig. 1):  GAL4-UAS, a transcription factor binding site....of PhiC31. In the SPARC system, attP sites are located on either side of a stop cassette, upstream of ...amount of labeling, you can alter the attP site. Truncating the attP site upstream of the stop cassette results...population is directly correlated to the length of the truncated attP site. The three SPARC variants, based on ...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ...Image from Isaacman-Beck et al., 2019. Applications for SPARC SPARC technology is versatile and can be ...

...into E. coli is a fairly inefficient process– just 1 out of 10,000 cells on average! Without some means...down especially fast and plates should be used witin 1 month for optimal efficiency. Beware of satellite ... perspective – a plasmid-containing cell must replicate the plasmid in addition to its own chromosomal...fully synthetic compounds. Antibiotics can be categorized based on whether they directly kill bacteria ...bacteriostatic); however, the distinction between the two categories may be a bit of a gray area as some bacteriostatic...Blocks 50S ribosomal subunit; inhibits aminoacyl translocation Bacteriostatic 50-100 ug/mL in EtOH Polymyxin...Binds 50S ribosomal subunit; inhibits peptidyl translocation Bacteriostatic 5-25 ug/mL in EtOH *In prokaryotes...

...and take a look at each of its parts. Figure 1: Parts of an AAV transfer plasmid. Parts shown in ..., ubiquitous expression, while the human synapsin 1 promoter is neuronal specific (Haery et al., 2019)...contains the proteins needed for the virus to replicate,  and the transfer plasmid which contains the ... by the AAV. On a plasmid map, its sequence is located between the ITRs. It can be anything you like as...expression of your transgene to a specific species or location. Promoters can also drive different levels of ...sequences that can flank the transgene and are located between the ITRs. Transfer plasmids often have ... the expression of your transgene. The WPRE is located at the end of the AAV genome, just upstream of ...

... similar antibodies in llamas and alpacas (Figure 1). At the time, researchers were already on a quest...nanobodies (Arbabi-Ghahroudi, 2017)!      Figure 1: Comparison of a heavy-chain only antibody and single-domain...composed of camels, llamas, and alpacas (and their undomesticated counterparts) - not exactly species that jump...to sdAbs becoming valuable tools for diverse applications. For example, RANbodies can be used as an alternative... excitement around using sdAbs in diagnostic applications. For example, fluorescently and radioactively...heavy-chain only antibodies. (A) Overview of genetic modifications made to generate the nanomouse. (VHH = variable...inspired this versatile family of tools, the applications they have already enabled, and the discoveries...

...more tools for plasmid mapping and design (Figure 1). Benchling: While you might think of Benchling as...Davis from the University of Utah.   Figure 1: A plasmid map created in SnapGene of Blue Flame plasmid...tackling these particular tasks, but they tend to be scattered about the internet. To help you keep your virtual...: Find sequences for plasmids assembled from publications and commercially available sources, as well ... as expression patterns, post-translational modifications and polymorphisms Codon optimization tool: To...making it ready to copy and paste into other applications Primer design tools Many of the above-listed... well as primer design for different cloning applications. Primer3 and Primer3Plus: For general primer...

...from distantly related organisms. In 2006, less than 1% of the genes deposited at Addgene were synthetic....synthetic biology is expanding rapidly.  References 1. Kunjapur, Aditya M., Philipp Pfingstag, and Neil ...published their results in a paper in Nature Communications announcing a new bioinformatics tool that can...at its sequence. MIT collaboration for gene classification This inquiry started when Neil Thompson, a ...have generated robust tools that have practical applications in monitoring the accidental or intentional ...from farther away in the tree of life." Nature communications 9.1 (2018): 4425. PubMed PMID: 30356044. PubMed...the lab-of-origin of engineered DNA." Nature communications 9.1 (2018): 3135. PubMed PMID: 30087331. PubMed...

...removing 1-20 nucleotides from the cleavage site specified by the gDNA. Depending on your application, this...About NgAgo User's First Impressions Here References 1. Gao, Feng, Xiao Z. Shen, Feng Jiang, Yongqiang Wu...template is supplied. Find Plasmids from the NgAgo Publication at Addgene Cas9 and NgAgo head to head NgAgo ...differences in cleavage efficiency. If this finding is replicated in larger studies, it will represent a key advantage...PubMed PMID: 27136078. Find plasmids from this publication at Addgene. 2. Swarts, Daan C., et al.  “DNA-... PMCID: PMC4697943. Find plasmids from this publication at Addgene. 3. Swarts, Daan C., et al. “Argonaute...

...organoid model (Sun, et al. 2021).  Figure 1: Example schematic from the Organoid EasyTag system...without altering that of the internal control (Fig 1). Because this method relies on the fluorescence ratio...be used to generate organoid cultures with a modification of interest. While the authors optimized the...mechanisms related to morphogenesis, cell fate specification, and human disease.     Sun, et al. bioRxiv...protoplasts, the enzymatic biosensor retains its catalytic activity.  As SLs are also known to trigger seed...parasitization of cereal crops. As a result, agricultural applications of these novel biosensors could have a major...

...B. Gurumurthy, and Masato Ohtsuka. Nat Protoc. 13(1) (2018):195-215. PubMed PMID: 29266098 Creating knock-in..., with protocols using dsDNA templates achieving ~1-10% efficiency. Easi-CRISPR uses a long ssDNA template...the gRNA design process and rules for various applications is necessary going forward. The standardization... I think I may have finally grasped just how complicated DNA repair pathways are. I fully admit to being...Alt-NHEJ), has been harnessed for CRISPR knock-in applications. CRISPR 101: Targeting RNA with Cas13a (C2c2...for CRISPR Genome Editing Other resources and publications CRISPOR gRNA design tool Researchers at the ...

...principles of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes... dopamine D2/D3 receptors. Scientific Reports, 10(1), 21842. https://doi.org/10.1038/s41598-020-78827-...for bacterial imaging . Microbiology Spectrum, 12(1), e0369023. https://doi.org/10.1128/spectrum.03690...Biochimica et Biophysica Acta - Biomembranes, 1838(1), 15–33. https://doi.org/10.1016/j.bbamem.2013.09.005...emission wavelengths, which are pivotal in their application in biotechnology (Grimm & Lavis, 2022). These... substrates/peptides and antibodies. Example applications of this include coupling to an antibody, whereby...whereby excitation of blue light will indicate where FITC is bound through green light emission. Key considerations...

...antisense RNAs inhibit plasmid replication.   Figure 1: The replication problem. Iterons are repeated...actually does, and neither plasmid is maintained (Figure 1).  The partitioning problem Plasmid incompatibility...Agrobacterium tumefaciens. Genes & Genetic Systems 77:1–9 . https://doi.org/10.1266/ggs.77.1 Pubmed PMID: ...Origin of Replication? See our Plasmids 101: Origin of Replication blog).  The replication problem Plasmids...directly inhibit replication by binding to the origin of replication and blocking replication machinery (del... of microbial plasmids: classification and identification based on replication and transfer systems and...plasmid that contains the origin of replication and the replication control machinery (Need a refresher...