...on a unique gene that makes the popular GatewayTM method possible: ccdB. But what is ccdB, what role does...it to evolve cloning technology. CcdB: A potent toxin... The ccdB gene, located on the F sex factor plasmid...the “swapping out” of ccdB with the investigator’s gene of interest.   CcdB resistant E. coli strains...toxic effects of CcdB. Another commercially available, CcdB-resistant strain is ccdB Survival™ from InvitrogenTM....1 or ccdB SurvivalTM, one can efficiently propagate and prep plasmids containing the ccdB gene, which... 2. Bahassi, EM., et al. “F plasmid CcdB killer protein: ccdB gene mutants coding for non-cytotoxic proteins...-selection vectors using the F plasmid ccdB killer gene”. Gene. 1994 Oct 11;148(1):71-4. PubMed PMID: ...

...recombination; ccdB gene for negative selection Used to clone attB-flanked genes of interest to generate entry ...E. coli strain sensitive to CcdB (e.g. DH5α, TOP10, Mach1). The ccdB gene is present in the donor vectors... clone and a byproduct containing the ccdB gene. Since the ccdB product is toxic to the cell, Gateway ... for recombination; ccdB gene for negative selection; elements to express the gene of interest in the ...attB sites is generated. As in the BP reaction, a DNA fragment containing the ccdB gene is excised from...attL sites. As a byproduct of the reaction, the ccdB gene is excised from the donor vector.   Figure... exchanged with the gene of interest during the BP or LR reactions. Since the CcdB protein inhibits the...

... the ccdB gene is replaced by the gene of interest. As a result, cells that retain the ccdB gene die off... of CcdB. Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is ...the ccdB region (for “coupled cell division”). The encoded toxin-antitoxin system consists of CcdB (toxin...is GatewayTM cloning, where the ccdB gene serves as a counterselectable marker in the destination vector...tool. The plasmid pKSCTMF contains the mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR...antisense RNA regulates hok gene expression indirectly through the overlapping mok gene." Journal of molecular...associated genes. These systems better ensure that plasmids are passed down through generations (Figure ...

...occurring genes for cloning purposes (antibiotic resistance genes, toxin genes, and the sacB gene). By tapping...destination plasmid, the ccdB gene is flanked by recombination sites. To introduce a gene of interest into the...plasmid excises the ccdB gene and the cells will only grow if they contain the gene of interest. SacB counter-selection...have lost the sacB gene. SacB can be used similarly to CcdB. For example, in the gene expression plasmid...gained a specific gene survive In positive selection, only cells that contain a specific gene survive. The ...selecting for the loss of a gene product - usually something toxic. This gene is found on the original plasmid...insertion of a DNA fragment within the gene or loss of the gene alleviates its toxic effect. Negative ...

... choice in assays ranging from use as a reporter gene in vitro and in vivo to use in biosensors. The most...effect of mutations of these regulatory elements on gene expression. Empty backbones for inserting regulatory...an intron in the middle of the Firefly luciferase gene.. TREAT (3( T hree)′- R NA E nd A ccumulation during...into which you can clone your regulatory element or gene of interest into to create a luciferase reporter... Firefly/Renilla Creating ORF-Firefly luciferase gene fusions. Renilla luciferase under the control of...enhancer regions. Mutations within the luciferase gene allow for increased copy number in E. coli and expression...cloning. Lentiviral. Jorge Ferrer 87067 pcDNA3.1-ccdB-Nanoluc NanoLuc® Creation of N-terminal Nanoluc ...

... for gene expression GFP Varies pBABE GFP - Retroviral gene expression pMKO.1...for mammalian gene silencing pLVTHM - lentiviral plasmid for mammalian gene silencing ...high expression of UAS-driven transgenes Worm unc-54, variety of worm gene promoters C. elegans vector ... nematode transgenesis Rosenberg Lab Plasmids - Transgenic single-copy gene ...part, we will assume that you want to express a gene; however, we have a section at the end for if ...If you want to drive expression of your favorite gene, you will need a plasmid with a promoter that...hybrid plasmids Fly UAS, MT Targeted Gene Expression - Rubin lab plasmids...

...Genotype ccdB Survival 2 T1R   TOP10 derivative. For propagating plasmids expressing the ccdB gene (important...derivative. For propagating plasmids expressing the ccdB gene (important in Gateway cloning). F- gyrA462 endA1...specific genes are signified with a minus sign (-) as is typically standard; just having the gene listed... of the more common genetic changes found in E. coli strains. Table 1: Common gene mutations found in ...carefully selected the optimal promoter for your gene of interest, painstakingly cloned into the perfect...backbone, made sure to add the right tags to your gene, and may have even put a fluorescent protein downstream...in E.coli strains Gene(s) Description Functional Consequence dam DNA adenine methylase mutation (GATC...

... plasmids containing the ccdB gene, such as Gateway vectors, choose the ccdB Survival strain. For plasmids...which growth strain Addgene should propagate your plasmids in: DH5α, NEB Stable, ccdB Survival, Pir1, or... this plasmid will work for them. Species of Gene or Insert Choose from: H. sapiens (human), M. ... Cloning Method Choose from: Gateway cloning, gene synthesis, Gibson cloning, ligation-independent ...information to Addgene Material Transfer Agreement (MTA) approval  Send plasmids to Addgene  Quality control... field, and contact Addgene. We may require that you send your samples to Addgene pre-transformed in this...the spreadsheet to deposit@addgene.org with the following information: Addgene account username. In order...

...Entrez Gene matches to your insert. Select the appropriate gene if it appears in the Entrez Gene list, ...plasmids containing the ccdB gene, such as Gateway vectors, we recommend the ccdB Survival strain. Figure...correct Entrez Gene does not appear in the auto-populated list, try entering the gene's Official Full ...known as" aliases in the Gene/Insert box. Once you have selected the appropriate gene from the list, save ...bacterial strain(s) through Addgene. Description – please indicate the gene cloned into your plasmid, any... for the gene or insert. Step 4: Growth and Distribution Please indicate which strain Addgene should propagate... Encodes insert(s) A plasmid with one or more genes, protein coding sequences, or other elements inserted...

... the ccdB gene containing BsaI restriction enzyme sites at its ends to generate pEcgRNA. The ccdB serves...multiple terminators can serve to interrupt genes (or gene clusters) in metabolic engineering. So far,...to successfully delete genes of interest in BL21(DE3) using the newly generated pEcCas/pEcgRNA system....used MUCICAT to optimize the gene dosage of glucose dehydrogenase and generated a strain that could achieve...self-curing, an arabinose inducible lambda-red gene, and an IPTG-inducible sgRNA sequence targeting the...non-temperature-sensitive replicon, and 3) added the sacB gene for counterselection to yield pEcCas. In pTargetF... of its E. coli host, and the addition of a sacB gene renders it beneficial for plasmid clearance. It ...

...coli strains, including DH5alpha, XL1 Blue, TOP10, ccdB survivalTM, and Stbl3TM. How to transform your plasmid...without antibiotics to give the antibiotic resistance gene time to be expressed.  However, you can skip this... perform over 100 transformations each week at Addgene, so we need to be cost and time efficient. We do...Preparation of electrocompetent cells (protocol) At Addgene, we use the Mix & Go! E. coli Transformation Kit...large number of transformations being performed at Addgene, this adds up to a significant time savings. You...

...the plasmid containing the lethal gene. An example is the ccdB gene system commonly used in Gateway® Cloning...are rescued by genes supplied on the transformed plasmid. Such plasmids may contain genes that allow for...auxotrophic cell lines), or genes which rescue a lethal phenotype. Resources on the Addgene Blog Read Our Post...well-characterized bacterial lac operon contains a gene called lacZ that encodes for the enzyme β-galactosidase...discovered that deleting a section from the lacZ gene (a mutation called lacZΔM15) creates a non-functional...other methods. Positive selection vectors encode a gene which, when expressed, is lethal to the cell. Cloning...fragments are inserted into an MCS in the center of this gene, disrupting the lethality. This is similar to α-...

...hosts through horizontal (or “lateral”) gene transfer. Horizontal gene transfer can occur naturally through...carrying genes for bacterial toxins like colicin typically also carry the necessary resistance genes to help...cloning relies on genes found on an E. coli fertility plasmid that also encodes a toxin, CcdB, and the resistance...Additional Resources on the Addgene Blog Plasmids 101: Antibiotic Resistance Genes Antibiotic Resistance: ...cell to another). Bacteria need special fertility genes to be capable of conjugation, and these are encoded...there is evidence that lightning can also promote gene transfer in soil bacteria! (Demanèche et al., 2001...organisms to thrive in competitive environments. The genes encoding these functions form the basis of many ...

...a single insert), mostly due to the use of toxic ccdB to prevent any non-recombinant vectors from propagating...technique, you first perform a round of PCR to generate DNA fragments with 15-40 bp of homology at their... site, toposisomerase I, and a taq polymerase-generated insert. When mixed, topoisomerase I provides both...Taq perfectly anneal with the topoisomerase I generated overhangs in the backbone. Once combined, the ...processes will require a PCR reaction or two to generate templates for the cloning process. Time spent ...spent actually doing the experiments are also generally comparable so the best way to compare costs is to ...assembly is a highly versatile and efficient way to generate a wide variety of constructs. Similar, Gateway...

...within the gene/insert, this information can be found here. There may be more than one gene inserted into...can easily clone my gene into it? Your best bet is the Plasmid Map: We auto-generate a plasmid map based... deposited with Addgene, you can list the original vector’s plasmid number here. Gene/insert: This describes...the backbone, and provides information about this gene or DNA element of interest. If the depositor has... into a backbone, and each is given its own gene/insert section on the plasmid page. Tags are listed according...strain DH5a, but others may require strains such as ccdB survival (for Gateway plasmids).  We also like to...We Do Quality Control at Addgene Resources on Addgene.org Join the Addgene Community Learn the Benefits...

...strains for storage and distribution at Addgene are DH5α, NEB stable, ccdb survival, and Pir1. The majority ...contain either an ampicillin or kanamycin resistance gene. Every step along the way involves good aseptic ...of tube for each plasmid we have at Addgene. Each sample at Addgene has four layers of location which can...pages! Resources Avaliable on the Addgene Blog Read more Inside Addgene Posts! Learn all about Plasmids ...Every plasmid sample enters Addgene the same way. A package is delivered by a mail courier and then the... a microcentrifuge tube. Once a sample enters Addgene’s lab, a series of events is triggered. Each step...fluctuates throughout the year (see graph below). Addgene scientists inspect each sample and make sure they...

...Gibson none S. pyogenes Zack pHL-H1-ccdB-mEF1a-RiH 60601 Mammalian BamHI/EcoRI none S. pyogenes Hygro Hotta...none S. pyogenes Mark pLenti-sgRNA-Lib 53121 Mammalian/Lentiviral BsmBI none S. pyogenes ccdB Wei pLKO5...are expressed as polycistronic glycine tRNA-gRNA genes (PTGs) and then PTG cassettes are assembled into...capable of simultaneously repressing multiple target genes in Escherichia coli. Koffas Do you have suggestions...BsaI none S. pyogenes Chloramphenicol Marraffini pCas9 42876 Bacteria BsaI yes, cut S. pyogenes Chloramphenicol...none S. pyogenes Qi pDD162 (Peft-3::Cas9 + Empty sgRNA) 47549 C. elegans yes, cut S. pyogenes Goldstein...elegans BsaI none S. pyogenes Joung pCFD3-dU6:3gRNA 49410 Drosophila BbsI none S. pyogenes Virmilion Bullock...

...contains a Gateway cassette containing the ccdB gene, a ccdB survival strain is necessary. What cell line...Available online: Genetic Organization and Genetic & Structural Features . In vivo gene delivery and stable...transfer plasmid. 2nd generation lentiviral plasmids utilize the viral LTR promoter for gene expression, whereas... rev, and tat genes all from a single packaging plasmid such as psPAX2 . 3rd generation packaging systems...packaging plasmid encoding the Gag, Pol, Rev, and Tat genes. The transfer plasmid contains the viral LTRs and...pictured). Unless an internal promoter is provided, gene expression is driven by the 5'LTR, which is a weak...: Uses 3 separate plasmids encoding various HIV genes. Safer. Replication incompetent and always SIN: ...

...effectively targets your gene/region of interest is a key step for CRISPR/Cas9 gene editing. Dr. Masahito...sequence or gene of interest into the pCoofy vectors, select the appropriate pair of vector and gene primers...vector and gene primer for SLIC cloning. Amplify the pCoofy vector and the sequence of gene of interest... can be used to generate random mutagenesis libraries or to insert heterologous genes, reporters, or other...Updated mini-transposon vector for bacterial mutagenesis or gene targeting Victor de Lorenzo's lab has...approach to genetic engineering which drastically reduces the time and effort needed to insert new genes into...successfully create GFP fusion proteins with a variety of genes across the genome. Although this tool was published...

...bacterial populations, an antibiotic resistance gene (i.e., a gene whose product confers resistance to ampicillin...transcription. Gene Knock-down Plasmids - Used for reducing the expression of an endogenous gene. This is frequently...chromosomes down to study their genes, marking the birth of molecular genetics. Today, scientists can easily...easily study and manipulate genes and other genetic elements using specifically engineered plasmids, commonly...when a gene is inserted within the MCS, its expression will be driven by the promoter. As a general rule...the gene, promoter, or other DNA fragment cloned into the MCS. The insert is typically the genetic element...experiment, see Addgene's empty backbone page for more information. Expression Plasmids - Used for gene expression...