What is the CcdB gene?
The ccdB gene, located on the F sex factor plasmid of E. coli, is part of a toxin-antitoxin system encoded by the ccd operon, which is responsible for plasmid maintenance during cell division.
What is the role of the CcdB gene in the donor and destination vectors?
The ccdB gene is present in the donor vectors and the destination vectors prior to recombination, and it is exchanged with the gene of interest during the BP or LR reactions. Since the CcdB protein inhibits the growth of CcdB sensitive E. coli strains, most colonies should contain the desired, recombined construct.
Is CcdB toxic to mammalian cells?
The particular property of the ccd system is that it targets the DNA gyrase, a topoisomerase absent from the cells of higher eukaryotes. The poison is therefore not toxic for mammalian cells.20. Positive selection of recombinant DNA by CcdB.
What does CCDB stand for?
CCDB
| Acronym | Definition |
|---|---|
| CCDB | Cell Centered Database |
| CCDB | Colorado Criminal Defense Bar |
| CCDB | Consolidated Counterdrug Database |
| CCDB | Credit Card Database (website) |
How does Golden Gate cloning work?
Golden Gate Cloning or Golden Gate assembly is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIS restriction enzymes and T4 DNA ligase. This assembly is performed in vitro.
What is an LR reaction?
The LR Reaction, again is a recombination reaction between attL and attR sites. The reaction generates an expression clone and is catalyzed by recombinant proteins. The entry clone generated from the BP reaction includes the attL sites. The Destination vector is designed to include the attR sites.
Is Golden Gate cloning scarless?
In practice, this means that Golden Gate Cloning is typically scarless. Additionally, because the final product does not have a Type IIS restriction enzyme recognition site, the correctly-ligated product cannot be cut again by the restriction enzyme, meaning the reaction is essentially irreversible.
What is scarless cloning?
Return to Cloning & Synthetic Biology. The group of cloning methods we refer to as “seamless cloning” typically combine attributes of more established cloning methods to create a unique solution to allow sequence-independent and scarless insertion of one or more fragments of DNA into a plasmid vector.
How does LR Clonase work?
In a Gateway LR reaction, an Entry clone is mixed with a Destination vector, and the LR clonase enzymes recombine the attL and attR sites of the matching subtype (i.e., attR4 with attL4, attR1 with attL1), swapping the Gateway cassette with the cloned insert.
What is MoClo?
Modular Cloning, or MoClo, is a relatively new assembly method introduced in 2011 by Ernst Weber et al., whereby using Type IIS restriction sites allows the user to ligate up to six DNA parts together in a one-pot reaction.
What is TOPO TA cloning?
TOPO cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′-end of the PCR products.
Is there a DNA gyrase resistant to CCDB?
One such strain is DB3.1, which contains a mutant version of DNA gyrase (gyrA462) that is resistant to the toxic effects of CcdB. Another commercially available, CcdB-resistant strain is ccdB Survival™ from Invitrogen TM.
Does restriction enzyme-mediated disruption of CcdB lead to identification of desired clones?
Figure 1: Image from Bernard, P., et al. showing the pKIL 18/19 vectors and demonstrating the concept of restriction enzyme-mediated disruption of ccdB, leading to positive identification of desired clones.
How does CCDB make cloning easier?
Read on to find out how ccdB can make your cloning experiments a little easier. One of the most time-consuming aspects of traditional cloning is the identification of clones that actually contain your insert of interest. Simply stated, ccdB makes cloning easier by selecting against vectors that did not take up your insert.
What is CCDB survival TM used for?
Using either DB3.1 or ccdB Survival TM, one can efficiently propagate and prep plasmids containing the ccdB gene, which can then be used for downstream cloning applications.