Applications for PCR cloning

The Staby®Cloning application is designed for the rapid, precise and efficient DNA cloning of PCR products. The complete cloning procedure is performed in one hour (including plating), the background is basically nil (the bacteria containing vectors without insert are killed), the PCR product is oriented, the plasmid is stabilized, and the export of the insert to another vector is easily selected.

Principle

The CYS22 bacterium used in this application contains a natural bacterial poison gene (encoding the selection protein CcdB) in its chromosome. This CcdB protein is only toxic in enterobacteriacae (E. coli, Salmonella). A truncated, inactive, antidote gene (ccdA) is present in the plasmid vector (pSTC1). This vector is linearized at the end of the truncated antidote gene. The ends of the vector are blunt. When a sequence of 14 base pairs (encoding the last 4 codons and the stop codon of the antidote gene) is added to the 5’-end of the DNA fragment to be cloned, the fusion of this sequence with the truncated gene restores an active antidote gene encoding an active protein (CcdA) able to counteract the action of the poison.

The 14-bp sequence is incorporated to the DNA fragment using one modified PCR primer. As illustrated below, this process allows the selection of (i) the recombinant plasmids that incorporate the fragment of interest, and (ii) the orientation of the fragment of interest (only one of the two possible orientations will restore an active, non-truncated, ccdA gene).

Results

Using the Staby®Cloning application, the complete cloning procedure is performed in 1 hour including plating. All the colonies plated are independent clones because no growth phase is required between transformation and plating. All growing bacteria contain the cloning vector with an insert in the correct orientation. Restoration of an active ccdA gene allows the selection of the recombinants and also stabilizes the plasmid into the bacterial population: if some bacteria lose the vector, they will die due to the action of the bacterial poison. After cloning of an insert, it is easy to export the stabilization cassette (antidote gene) together with the insert using the restriction sites in the vector. The insertion into another vector can be selected using the CYS22 bacteria and the antidote properties.

Benefits

  • SPEED: Ligation + transformation + plating in one hour;
  • PRECISION: Vectors without insert are eliminated (background<1%) and the DNA fragment is correctly oriented in the vector;
  • MOBILITY: Export of [stabilization cassette+insert] is easily selected into other vectors;
  • Recombinants are all independent clones (direct plating);
  • No “satellite” colonies observed even after extended incubation;
  • No risk of background when using AmpR plasmids as PCR template;
  • Recombinant plasmids are perfectly stabilized without the use of antibiotics;
  • The system is usable in any culture medium.

The Staby®SubCloning application was designed to transfer  genes of interest from Staby®Cloning vector (pSTC1) to efficient expression vectors. This transfer is very easy and powerful: using simple restriction sites and ligation, your insert is oriented and selected in the appropriate sub-cloning vector, it is not required to purify your insert.The multiple cloning sites of destination vectors contain the same compatible restriction sites. Consequently, one restriction of the pSCT1 vector is compatible with all Staby®SubCloning vectors.

During the cloning step of a PCR product in the Staby®Cloning vector, the gene of interest is oriented and linked to the ccdA antidote gene (see the Staby®Cloning section). Using restriction sites in the pSTC1 vector, it is easy to transfer the gene of interest (linked to the ccdA antidote gene) into a sub-cloning vector. The ccdA presence linked to your gene allows selection of recombinants in CYS22 or SE1 bacteria (these bacteria contain a natural bacterial poison gene (encoding the poison protein CcdB counteracted by CcdA) into their chromosomes). Since this selection is very efficient, it is not required to purify your insert linked to ccdA gene before sub-cloning. The restriction-sites choice allows selection of insert orientation. After sub-cloning, the vector is ready for protein expression in SE1 bacteria.

The restriction sites were chosen to be compatible with sites of pSTC1 vector and to place your gene in frame with the protein tags. Several sub-cloning vectors were developed for each application: protein expression with his-tag, no tag, cherry tag, missing t-RNAs (see Staby®Codon), yeast protein expression...

The multiple cloning sites of destination vectors contain the same compatible restriction sites. Consequently, one restriction of the pSCT1 vector is compatible with all Staby®SubCloning vectors.

Vector maps:

Available on request

-pSSC-Native1
Features:T7 promoter, no tag, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS22 and SE1 bacteria after sub-cloning.

-pSSC-His1
Features: T7 promoter, His tag, Enterokinase cleavage site, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS22 and SE1 bacteria after sub-cloning.

-pSSC-Cherry1
Features: T7 promoter, Cherry tag, Enterokinase cleavage site, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS22 and SE1 bacteria after sub-cloning.

-pSSC-Native2
Features: T7 promoter, no tag, additional t-RNAs, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS22 and SE1 bacteria after sub-cloning.

-pSSC-His2
Features: T7 promoter, His tag, Enterokinase cleavage site, additional t-RNAs, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS22 and SE1 bacteria after sub-cloning.

-pSSC-Cherry2
Features: T7 promoter, Cherry tag, Enterokinase cleavage site, additional t-RNAs, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS22 and SE1 bacteria after sub-cloning.