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We narrowed to 23 results for: ccdb gene

Showing: 1 - 20 of 23 results

  1. Plasmids 101: CcdB - The Toxic Key to Efficient Cloning

    Type
    Blog Post
    ...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: ...

  2. Plasmids 101: Gateway Cloning

    Type
    Blog Post
    ...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...

  3. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    ... 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 ...

  4. Plasmids 101: Positive and Negative Selection for Plasmid Cloning

    Type
    Blog Post
    ...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 ...

  5. Plasmids 101: Common Lab E. coli Strains

    Type
    Blog Post
    ...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...

  6. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ... 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...

  7. Luciferase Plasmid Collection

    Type
    Collection
    ... of your gene of interest upstream of IRES-firefly luciferase. Bryan Welm 87067 pcDNA3.1-ccdB-Nanoluc ...Explore Addgene's collection of luciferase plasmids for gene expression assays and bioluminescent reporters...luciferase-containing plasmid and the effects on gene expression can be measured by the luminescence output...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... Retroviral expression of firefly luciferase and gene of interest Scott Lowe 21375 pHIV-Luciferase Firefly...

  8. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    ...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...

  9. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    ... 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 ...

  10. Save Time and Money by Making Your Own Competent Cells

    Type
    Blog Post
    ...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...

  11. Plasmids 101: Blue-white Screening

    Type
    Blog Post
    ...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-...

  12. Plasmids 101: The Wide World of Natural Plasmids

    Type
    Blog Post
    ...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 ...

  13. Plasmids 101: 5 factors to help you choose the right cloning method

    Type
    Blog Post
    ...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...

  14. Anatomy of a Plasmid Page at Addgene

    Type
    Blog Post
    ...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...

  15. Tips from the Repository Trenches: Using Barcodes to Track Samples

    Type
    Blog Post
    ...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...

  16. CRISPR Plasmids - Empty gRNA Vectors

    Type
    Collection
    ...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...

  17. Empty Backbones - Choosing Your Perfect Plasmid Backbone

    Type
    Collection
    ... vector for gene expression pINDUCER21 (ORF-EG) - Inducible lentiviral gene expression ...destination vector for gene expression Lentiviral backbones made by Addgene Addgene has an extensive collection...host cell and are designed for a user to insert a gene of interest. They may contain tags or fusion proteins...These vectors allow you to test the function of your gene of interest in a controlled environment under various...we will assume that you want to express a gene; however, we have included a function section for if you...If you want to drive expression of your favorite gene, you will need a plasmid with a promoter that...mTagBFP2 tag pNIC-GST-TEV-GG - Cloning of target gene with N-terminal GST tag and TEV cleavage Transient...

  18. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...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...

  19. Gamma-Retroviral Vector Guide

    Type
    Guide
    ...contains a Gateway cassette containing the ccdB gene, a ccdB-resistant strain is necessary. For more information...the necessary packaging genes, while lentiviruses also contain accessory genes specific to each virus ...common packaging genes gag , pol , and env . The LTRs flank all of these required genes, and anything in...— contains packaging genes gag and pol Envelope plasmid — contains packaging gene env ; usually VSV-G ... envelope gene, a process called pseudotyping. VSV-G is by far the most common envelope gene, as it has...commonly used in gene therapy applications to more specifically direct delivery of gene therapies. Common...gamma-retroviral vectors. Gene Therapy Gamma-retroviral vectors are a popular choice in gene therapy applications...

  20. Adenovirus Guide

    Type
    Guide
    ...contains a Gateway cassette containing the ccdB gene, a ccdB-resistant strain is necessary. For more information...provide a large transgene packaging capacity. Large protein-coding genes and complex gene expression cassettes...(2017). Adenovirus-mediated gene delivery: potential applications for gene and cell-based therapies in..., subsequent generations of rAdV vectors have been developed where more adenoviral genes have been removed...containing the adenoviral genes necessary for viral production. In AdEasy™, the transgene of interest is cloned...containing the transgene of interest, and the adenoviral plasmid containing the adenoviral genes necessary ...second and third-generation vectors. This capacity is large enough to carry the Cas9 gene and a gRNA expression...
Showing: 1 - 20 of 23 results