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...mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct...than plasmids without this DNA. They eventually designated this fragment as the ccdB region (for “coupled... serves as a counterselectable marker in the destination vector. Successful cloning occurs when the ccdB...restriction endonucleases in plasmid addition was first described by studies of EcoRI and Bsp6I in Escherichia coli...systems. As with the other toxin-antitoxin systems described above, one part of the pair is more stable than...toxin-antitoxin system discovered. Unlike the systems just described, the hok/sok systems consist of three components...long-term experiments. They’ve also helped researchers design and optimize cloning methodologies using counterselection...

... vivo and in vitro applications.   Figure 2: Modular features of DIRECTED particles where the ...unstructured templates and significantly reduced undesired edits (indels, etc.), while PE6c and PE6d were...Delivery to Intended REcipient Cells Through Envelope Design). This modular approach combines an interchangeable...Cell type-specific delivery by modular envelope design. Nature Communications, 14(1), 5141. https://doi.org...LOV-Turbo plasmids here! Next, Qin and Cheah et al. described TransitID, where two proximity labeling enzymes...that had been trafficked from the “source” to “destination”. Images adapted from Lee and Cheah et al. 2023...Figure 4: Time-resolved protein ticker tape. A) Design of iPAK4 protein ticker tape recording. B) HEK ...

...based antiviral method to degrade RNA from SARS-CoV-2 sequences and live influenza A virus. It was demonstrated...their BioRxiv post, the authors generated and described several improved genetically encoded fluorescent...Takayuki Iwaki, Kimiko Hara, and Kazuo Umemura have described a simple method which alleviates the inclusion...contains the signal peptide of MBP, followed by the desired nanobody (NB) sequence, an AviTAG, and 6xHis tag...expression can vary significantly depending on transgene design and delivery. To overcome these variations in human...Addgene The MoClo CRISPR/Cas Toolkit for plants includes 95 plasmids consisting of CRISPR/Cas nucleases... uses CRISPRi to control gene expression. This includes a CRISPRi-based synthetic oscillator, bistable...

...PMID: 18478103. PubMed Central PMCID: PMC2364656. 2. Dittmar, Kimberly A., et al. "Selective charging ...features to consider when designing a full gene. A non-exhaustive list includes: Codon abundance relative...that their codons are more appropriate for the desired expression host. This is known as “codon optimization...as easy as it sounds. Even for relatively short peptides, there can be many possible ways to encode them...every amino acid I’d like to encode,” but, as described above, not every region of a protein should necessarily...PubMed Central PMCID: PMC4794256. This review provides a great overview of codon usage bias 8. Tuller...

...the necessary steps to perform:      Fig. 2: Quick-stepping to a FASTA file   With our outline...of data. In this two-part blog series, we will describe the importance of bioinformatics at Addgene and...each week. Our internal software was originally designed to help us work in large formats, such as 96-well...Thanks to the flexibility inherent in Nextflow’s design, we were free to use multiple programming languages...alone! The benefits of containers are they are designed to ‘contain,’ in a complete package, all of the... who or which computer ran the process. Another design choice we appreciated about Nextflow was how effortless...small with a print statement and tested that the desired data was available so we felt confident we were...

...transposase systems utilize with type I-F Cascades. Figure 2: Cascade complex from CRISPR type I-E....Cas11. Type I-E Cascades contains all five of these Cas proteins, while type I-C Cascades do not contain... 1: Schematic of Class 1 CRISPR types. Class 1 includes type I with a Cas3 effector, type III with a Cas10...maximum efficiency of the process. Various labs have designed all-in-one Cascade plasmids — such as the Tomoji...

...rate of 10-55% - the PASTE system.     Fig. 2: Schematic of programmable gene insertion with PASTE...The hardest part of any revolutionary discovery, including CRISPR, is turning potential into impact. ...two pieces: a single protein, computationally described as a fusion of four Cas7 proteins and a putative...RNA editing methods — streamlining experimental design and its potential for clinical applications. Specifically...high-fidelity rate. Both Cas7-11 and PASTE open up multitudes of possibilities at the bench and potentially...

...that view (Callahan et al., 2022).     Fig. 2: Students in an MDH CURE replaced select residues ...semester one and then proceed to use those skills to design and carry out their own novel experiments in semester...resources, offers mentoring for instructors, and provides opportunities for students to exchange ideas and...results in student learning gains and positive STEM attitudes in CUREs. CBE Life Sciences Education. 21(4):ar74...

...would help them grasp some of the key concepts (Fig. 2). One thing I will likely change for next time is ... can teach  5-10 year olds in a single evening. Grades K-5 cover a broad range of attention spans, skill...CRISPR work? This section was essentially background slides getting everyone on the same page in terms of the... by running it by the teachers and sharing some slides on Twitter, where I was able to get some great ...

...replicate, then purify the DNA.     Figure 2: A pooled library page with protocols boxed in red... If not, our protocol on Lentivirus Production provides a general approach for producing lentivirus in...excellent protocol paper by the Feng Zhang lab describing construction, lentiviral production, transduction...the Broad Institute- A variety of protocols and guides regarding pooled libraries, from cloning a pooled...

...pairs.  Figure 2: (A) The codon wheel depicts the 64 codons that encodes the 20 amino acids and...Usage Tabulated from GenBank database (CUTG), that provides an electronic dataset for codon-usage of 257,468...Codon Optimization  ATUM DNA2.0  IDT Synthetic gene design made easy GenScript- GenSmart Codon Optimization...

...PMID: 24901218. PubMed Central PMCID: PMC4047077. 2. Pagliara, Patrizia, et al. "Ostreopsis cf. ovata ...and could be contaminated.Learn how Addgene uses barcodes to track samples Keep track of passage numbers...

...experience and to build upon your existing skills.  2. Find your niche Identify and develop an area of expertise...LLC, a grant professional development firm that includes the social media community GrantChat. She has ... from administration and fundraising to program design and evaluation. She and her team at J. Miller &...

...externally fertilized, transparent progeny (Figure 2). These features give it a leg up (or a fin up?) over...deactivation method in zebrafish are short, modified oligonucleotides called morpholinos. When injected into zygotes...CRISPR/Cas9-mediated insertion of foreign DNA. Despite these caveats, several features of zebrafish—including...

...effective recovery of knock-ins, but did result in 1-2 kilobases of additional sequence being introduced....https://doi.org/10.1093/nar/gku289 Chen C, Fenk LA, de Bono M (2013) Efficient genome editing in Caenorhabditis... DNA double-strand breaks up to 54 bp from the desired insertion site result in efficient editing. Repair...available through Addgene. In 2019, Farboud et al. described another similar co-conversion marker (repair of...whether it be by improved gRNA or repair template design, delivery, screening, and more. Here are some highlights...highlights from the past few years:  Repair template design The Meyer lab meticulously characterized several...of editing. First, they devised guidelines for designing single-stranded repair templates (Farboud et al...

...work. Nobody wins, and you need to make a change. #2 Thou shalt sometimes put work ahead of life Sometimes...that extra two miles which is way more fun. And despite those who may wish to tell you so – towel corners...

... Watch Joanne give her "Not" Networking 101 Talk 2. Tailor every talk to the occasion Don’t just plan...suspense. Notice the simplicity of their slides and how the slides complement the spoken message. Learn from...speak to start creating slides or thinking about content. Create a Google.slides or Powerpoint file right...how these speakers talk without looking at their slides or notes. Listen to their stories and narratives...listeners to be thinking “What happens next?”  Describe how you pushed to a solution or overcame obstacles...term, but are worried that this might sound condescending, consider explaining the term on a slide but...talk will really take and to adjust content and slides to better suit the target time frame.   I suggest...

...of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO), a fluorophore modified from DsRed. DFHO...Aptamer Fluorophores describes the enrichment process used to evaluate oligonucleotides for binding affinity...of Spinach. In doing so, they identified the nucleotides that contribute to the instability of Spinach...similar levels of fluorescence when bound to DFHBI despite being approximately half the length (Warner et ...also smaller than any form of Spinach - only 49 nucleotides in length - and is better able to maintain its... of colors, so do aptamers! Corn was recently described in 2017 (Song et al., 2017). Corn naturally dimerizes... Red and orange fluorescent aptamers Corn was described in the same study as Red Broccoli and Orange Broccoli...

...PubMed PMID: 3459152. PubMed Central PMCID: PMC323600. 2. Chester, Nicholas, and Daniel R. Marshak. "Dimethyl...for the desired modification. Finally, the positive clones are sequenced to confirm the desired modification...Primer Design As a rule of thumb, 11 bp of complementary sequence on either side of the desired mutation...introduced to plasmids using primers (with the desired mutation) in a PCR protocol that amplifies the ...mutated clones. Forward and reverse primers are designed to be complementary, but each primer may extend...Polymerase To ensure that you don’t introduce undesired mutations through the PCR process, you need a ...The resistance marker from the parental plasmid provides a mean for selecting for transformants which have...

...gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) General protein expression...and some of the most popular commercial strains designed to avoid them. Why do I need an expression strain...protease: Strains where this is completely deleted (designated lon or Δlon) similary reduce proteolysis of the...which can prevent the culture from reaching the desired density within a reasonable time-frame. For these...and pLysE—the difference being that the latter provides tighter control of basal expression. What if I...don't see protein overexpression? The strains described above should generate sufficient expression levels...you’ve tried a common strain and don’t get the desired level (or any) protein expression? Low expression...