What is CcdB survival?

Description. One Shot® ccdB Survival™ 2 T1R Chemically Competent Cells are suitable for the propagation of plasmids containing the ccdB gene, and are designed for use with the Gateway® Vector Conversion System and for propagating Gateway® destination, donor, and supercoiled entry vectors.

Is CcdB gene 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 is CcdB resistant E. coli?

CcdB resistant E. coli that tolerate the expression of the toxin gene. One such strain is DB3. 1, which contains a mutant version of DNA gyrase (gyrA462) that is resistant to the toxic effects of CcdB.

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 DH5alpha ccdB resistant?

This was a good answer but there is evidence that the gyrA96 (NalR) mutation in common cloning strains such as XL1blue and DH5alpha does impart resistance to ccdB.

What is F Episome?

Nomenclature. Episome: A circular piece of DNA that can replicate independently of the bacterial chromosome or integrate and replicate as part of the chromosome. F factor: An episome in bacterial cells that confers the ability to act as a genetic donor during conjugation.

What is SnapGene?

SnapGene is molecular biology software that allows users to plan, visualize, and document molecular biology procedures. Select the DNA fragments that you wish to fuse, and SnapGene will design the primers. SnapGene Viewer is software that allows you to create, browse, and share richly annotated DNA sequence files.

Is Gateway cloning expensive?

Using Gateway, one can clone subclone DNA segments for functional analysis. Gateway cloning does take more time for initial set-up, and is more expensive than traditional restriction enzyme and ligase-based cloning methods, but it saves time, and offers simpler and highly efficient cloning for down-stream applications.

What is entry clone?

An Entry clone contains your gene of interest flanked by attL sequences, which are then used to recombine with attR sequences to create your desired expression clone.

What is the difference between DH5 alpha and BL21?

The key difference between BL21 and DH5 Alpha is that BL21 is a protease deficient genetically engineered competent E. coli cell used primarily for protein expression, while DH5 Alpha is a genetically engineered competent E. coli cell with recA1 mutation used primarily for plasmid transformation.

Is DH5 Alpha ampicillin resistance?

Yes, DH5 alpha carries no resistance to antibiotics. It means it should be manipulated very carefully, using stringent asseptic conditions.

Can f+ conjugate with F+?

F+ or Hfr+ strains could no longer be a recipient to mate.

How does CCDA protect the cell from CCDB?

ccdA, another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the cell against the toxic CcdB. Cells that lose ccdA through the loss of the F plasmid, succumb to the toxicity of CcdB.

What are the vectors that contain the ccdB gene?

These vectors, called pKIL18 and pKIL19, contained the ccdB gene in frame with an MCS. E. coli that were transformed with the empty vectors expressed the ccdB gene and were therefore unable to propagate because CcdA wasn’t available to counteract the toxin.

Which is the toxic gene in the ccd operon?

ccdB codes for the toxic protein (CcdB) that acts as a DNA gyrase poison, locking up DNA gyrase with broken double stranded DNA and ultimately causing cell death. ccdA, another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the cell against the toxic CcdB.

Are there any plasmids that are 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.