...Host range depends on whether the host has the components to recognize the ori and allow for replication...organisms. pBBR1-based plasmids and plasmids in incompatibility groups N, P, Q, and W are known to have replicons...list of the bacterial species these plasmids are compatible with.  Table 1: Non-exhaustive list of common... and Srivastava 2013. Family Replicon Compatible with Shown to work in IncN pCU1 Gram-negative...

...meets two conditions:  1) The sequence is unique compared to the rest of the genome.  2) The target is present...found. If you’re lucky enough to find a SpCas9 compatible PAM at your site of interest, that is the most...expanding number of Cas9s with looser PAM requirements compared to SpCas9. xCas9 has a PAM requirement of NG, ... proteins. The Synergistic Activation Mediator complex, generally the strongest activator, consists of...

...can be difficult to construct and deliver when compared to gRNAs. Additionaly, cDNAs cannot be used to...Housden BE, Perrimon N, Collins JJ, Church G (2016) Comparison of Cas9 activators in multiple species. Nat Methods...transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517:583–588 . https://doi.org/10.1038/nature14136...Kadoch C (2018) Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale...

...to the popularity of GFP including (i) fast and complete maturation to functional, fluorescent protein ...diameter of about 2.4 nm (Hink et al., 2000). The complete beta-barrel is necessary for its fluorescence ...genetically encoded tag and exogenously added components. For a review see Lotze et al. (2016). To date...is used to excite GFP, may also excite several components that are naturally present in cells or media....

...endogenous homologous recombination enzymes can also accomplish the desired genomic modification. This technique... which results in a merodiploid. This can be accomplished by selecting for an antibiotic resistance marker...scarless modification of bacterial genomes. It can accomplish large deletions, insertions, and even precise...PMC4513841. Latynski US, Valentovich LN. "DNA tuner: a computer program for the construction of polylinker sequences...

... proteins in living cells and organisms. Both components of the chimera are encoded by DNA. Since researchers...When both orientations are available, a direct comparison of these fusion proteins may reveal which of ...the fusion protein can be verified and it can be compared to what is expected based on immunofluorescence...interactions) can be challenging. If a fusion can complement a knock-down or knock-out of the corresponding...

... (Sandomenico et al., 2020). Figure 1: Comparison between the IgG antibody and scFv.   What...their lack of an Fc region. Disadvantages scFVs:  Compared to antibodies, scFvs tend to have lower affinities...intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged with ....0000000000000145  Gąciarz A, Ruddock LW (2017) Complementarity determining regions and frameworks contribute...

...the production of lentiviral vectors that are competent for one round of infection (AKA transduction in...progeny are produced (Figure 1).  Figure 1: Comparison of the life cycle of a lentiviral vector with...double-stranded DNA transgene is incorporated into a large complex of both viral and cellular proteins and trafficked...titering IDLVs include detecting vector particle components or performing quantitative PCR titration of vector...

...method for knockouts is Tracking of Indels by Decomposition (TIDE) (Brinkman et al., NAR). This technique...trace files of WT and edited cell lines. Then decomposition analysis is performed yielding a graph of edits...This method requires a set of control cells to compare the sequencing reads from your edited sample to...assessment of genome editing by sequence trace decomposition. (2014) Nucleic Acids Research Volume 42, Issue...

...lacY1 proA2 rpsL20 xyl-5 leu mtl-1 Stbl4   Electrocompetent cells for cloning and storage of unstable ...XL10 Gold Tetracycline and Chloramphenicol High competency cloning and propagation of large plasmids, ligated...tables are by no means exhaustive! For a more comprehensive list and additional information, visit OpenWetWare's...of Bacterial Expression Systems. Check out our companion post describing protein expression strains, where...

...helps all viewers, especially if you have some complex patterns with several overlapping targets to interpret...you add the secondary antibody, it should get completely washed away and leave no signal. If you see lots...samples where you label only one target in each. Compare these individually-labeled samples with the results...deep phenotyping and spatial analysis of cells in complex tissues. Proceedings of the National Academy of...

...certain Cas enzymes are less compatible with the bulkiness that accompanies mispairing of bases. To reduce...unnecessary for many applications, it is the only comprehensive method available. Targeted sequencing approaches...must be quantified slightly differently. For a comprehensive approach, RNA-seq would be an appropriate method...

...could avoid potential off target artifacts by comparing the phenotypes of multiple gRNAs. Targeting different...lanes 0.3 % 3.0, Figure 5, lane 2) is faint, so comparing it to the undigested control (Figure 4, lane 0...instead of agarose gives much sharper bands. I compared two MOIs of gRNA transduction to see if that had...testament to how far genome engineering has come. For comparison, in 2007 it took about 3 months to knockout a...

...your data by Nov. 12 to compete. The top ten participants will be invited to compete in our speed round data.... Bobae An will host a Q&A session after the competition, where you can get all your burning AAV questions...

...In complex metazoans, rapid cell division and large scale cell mobility are essential processes during... required for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...organism. Studying cell mobility within these complex environments has never been easy and, for many ...labelling of cells using cre-lox is advantageous when compared to photoactivable or photoconvertible proteins...

...straightforward to learn and relatively cheap (compared to many other technologies and techniques in the...experiment work? What are the various tools and components necessary to run it? And, most importantly, what...writing. Derek is also a pianist and an aspiring composer. View his blog drsimonsaysscience.org or follow...applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nature methods....

...fragments, allowing ordered assembly of multiple components into a destination vector. The first step in ...Oligonucleotides specifying the gRNA seed sequence should be compatible with type IIS enzyme BfuAI. Gateway cloning is...supply it with another plasmid.   Figure 3: Comparison of Multiplex Strategies including Standard PolIII-gRNA...Cas9-containing binary vectors. The system is compatible with both monocot and dicot plants. Liu Lab Golden...

...easier to generate mouse models.   Cpf1 Update: Comparison to Cas9 and NgAgo As you might expect given the...off-target editing and similar on-target editing when compared to the most commonly used Cas9 variant, SpCas9...Generating Mouse Models Using CRISPR/Cas9 Cpf1 Update: Comparison to Cas9 and NgAgo Special Delivery: Fluorophore...

...are of interest to you, our readers. Below I’ve compiled a list of the Top 10 Most Viewed Posts on our ...learn more about. Our post on “Making Your Own Competent Cells” has been extremely popular, generating ...Morgan Save Time and Money by Making Your Own Competent Cells By Melina Fan Plasmids 101: The Promoter...

...difficult to compare the performance of different nucleases. Nicola Patron’s lab compared the efficiency...more predictable and precise deletion products compared to monomeric Cas9’s. Read the paper in Nature...

...’d like to take a moment to reflect on our accomplishments from 2022 and share what we’re looking forward...ready-to-use recombinant antibodies collection, currently composed of over 100 antibodies. Everything in this collection...upon, a wealth of history to learn from, and a compelling future to plan in the changing landscape of science...

...tags with defined 3D structures less likely to compromise protein folding and function. Using bioinformatic...GFP did not reduce yeast cell size or viability compared to untagged GFP overexpression, while the rest...cell size and growth rate, indicating that they compromised protein-protein interactions. If you’re looking...

...research and training is getting tighter and competition is fierce. However, I believe there are plenty...Washington University founded a successful consulting company called The Balsa Group. University of Pennsylvania... Consulting. I know of many schools that have completely student/postdoc-driven biotechnology clubs such...

...cassettes for selection of transgenic plants. The complete GreenGate toolkit, now available at Addgene, also...GreenGate can do is “save you half a year,” in comparison to more tedious and time-consuming traditional...traditional cloning methods, Lohmann said. The key component of Golden Gate and GreenGate are type IIS restriction...

... For more details, check out this comprehensive review comparing optogenetic CRISPR techniques (Matony...repeatability. The system developed by the Sata lab is composed of two fusion proteins: 1) the genomic anchor ...of transferrin receptor CD71 in HeLa cells.   Comparison of photoactivatable CRISPR strategies Publication...

...labels are obtained by independent audit, and companies need to pay to obtain them. There’s a database...you don’t see one, be an ACTivist and encourage companies to obtain these labels! Reusing plastic Historically...benchtop, high throughput washing devices that are compatible with a variety of pipette tips. This is part ...

...bird feces, which get eaten by another snail, completing its lifecycle. Halloween genes The Halloween ...grasshoppers expressed different proteins in their brains compared to uninfected grasshoppers, including those linked...Additional resources on the Addgene blog Read about company culture at Addgene See some of our previous Halloween...

...could take all of the credit, the success of the company is attributed to the entire Addgene family; the... on sliders, hot dogs and a mashed potato bar (complete with all the fixings), as well as plenty of delicious...course of scientific research. What was once a complicated process of requesting and sharing plasmids has...

... Blue light renders the NLS accessible and the complex enters the nucleus where dCas9-Mxi1 can repress.... In the dark, the NLS is inaccessible and the complex remains in the cytoplasm. Read the paper in Cellular...CRISPR-based DNA demethylation Figure 2: The components of Casilio-ME1. Image from Taghbalout et al.,...

... come in the new year. I am proud of what we accomplished in 2023 and thrilled to be sharing our plans...addition, we released the MORF collection, a comprehensive transcription factor library, in lentiviral ... efficiencies typically adopted by for-profit companies, while making use of our nonprofit status to achieve...

...information.” Addgene is here to help scientists accomplish their goals. Many nonprofits must spend a lot...level of positive revenue to build reserves so the company is sustainable. The difference is what nonprofits...-oriented values. Addgenies like the nonprofit company culture as well. We work every day to do a better...

... expert (SME) at eScience Labs, an educational company that creates hands-on science lab kits and digital...with faculty members to make sure the courses encompass what they want to teach.” Besides working with...lab: write!” Caitlin did this by editing 15+ comprehensive exam proposals, a few PhD theses, and occasionally...

...of this effort, members of the Rawlins Lab have compiled a new toolkit for performing genetics studies ...workflow combines thoughtfully designed plasmid components with Fluorescence Activated Cell Sorting (FACS...in high throughput screening for agrochemical compounds that have the potential to regulate the parasitization...

...change in pathology. If the disease pathology is complex, a model may be most effective. Once drug candidates...and generate physiologically relevant models. In complex diseases, it can aid in generation of multiple ...specificity of that target and validate the hit compound. The standard is to test the drug on models which...

...through biomedical research. The Camelid family is composed of camels, llamas, and alpacas (and their undomesticated...Arbabi-Ghahroudi, 2017)!      Figure 1: Comparison of a heavy-chain only antibody and single-domain...blog over the years and have described how they compare to other antibodies and affinity reagents. But ...

...transfer in vertebrates. The system is comprised of 2 components: 1) an SB transposase, the enzyme required...efficiencies of stable gene transfer using SB100x were comparable to stable transduction efficiencies of integrating...

...CRISPR delivery systems, modifications to the RNA components, and challenging target genomes, such as the ...foremost, from the custom software used to design complex genetic circuitry to the downstream output. Tuesday...digital and analog versions of each, and Tim Lu even compared his "memory" gene codes to a Turing machine (also...

...labs today are semi-synthetic or fully synthetic compounds. Antibiotics can be categorized based on whether...gene of interest is cloned into/inactivates or completely removes (in the case of Gateway cloning) one ...make sure your plasmid and E. coli strain are compatible! Check out this list of common E. coli genotypes...

...faithfully transcribes RNA to DNA with low error rates compared to other RTs. MarathonRT represents a major step...long-read cDNA sequencing and RNA sequencing of complex heterogeneous RNA samples. Furthermore, such high... restricted spatial tagging), to confirm the composition of proteins found at the telomere, and hope it...

...common lexicon for genome editing studies. These complementary goals are aimed at improving translation of ... loosely paraphrased in the tweet below. The complexity and challenges of homology directed repair (HDR... I think I may have finally grasped just how complicated DNA repair pathways are. I fully admit to being...