Team:Uppsala University/Backbones

There have been longstanding doubts about the behaviour of the existing pSB4x5 series of low copy plasmids. Due to a need for a low copy BioBrick plasmid in our project, we have devoloped a series of BioBrick standard vectors. The new pSB4x15 series have a low copy pSC101 replication origin (~5 copies per cell) and ampicillin, chloramphenicol, kanamycin and spectinomycin antibiotic resistance markers. They are especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22, similar to BBa_K59200.

The pSB4x15 series i brief

• Verified low copy number
• More accurate annotation
• No homologies to E coli genome that interferes with Lambda red recombineering
• Easy introduction of new resistance casettes
• Easy introduction of new orthonongal replication origins
• Smaller backbone size
• Flp recombinase target sites aroud the resistance casette available
• LacIq versions for tight represion available
• Thermosensitive version available

General design notes
The resistance cassette is terminated by a Lux bidirectional terminator. Adjacent to this terminator there is a designed primer binding site to where the forward primer for lambda red can be designed to bind to, regardless of insert in the cloning site or resistance. For more information on Lambda Red recombineering, read in the description for part BBa_K59200.

The resistance cassette is flanked by SalI and SacI restriction sites for easy switching of resistance in the backbone. In the case of the Frt plasmids, there is a Flp recombinase target site inside of each of the flanking restriction sites, for removal of the resistance cassette after chromosomal integration. The Frt plasmids can, however, also be used as normal cloning vectors.

The pSC101 origin of replication is flaked by NheI and MluI restriction sites for easy switching of origin of replication. An illegal SpeI restiction site has been removed from the origin.

Nomenclature

• pUCori - a high copy origin of replication. Amplified from the pSB1A3 backbone.
• red - the standard PlacI-RFP casette J04450.
• redq - a RFP casette driven by the CP44 constitutive promoter [1], but otherwise identical to J04450.
• There are also a composite parts of pUCori-red and -redq, K864120 and K864121.

For expression of toxic genes, or simply genes where you want to be able to tune the expression level, we constructed a series of lacIq bacbones. Including the lacIq casette on the plasmid ensures that the copy number of the repression always follows that of your inserted genes, providing guranteed strong repression without inducing unneccessary metabolic load.

The pSB8 backbones contain the pSC101ts ori which, due to a change from alanine to valine in a regulatory protein[2], does not replicate at 42°C[3]. Strains carrying pSB8x15 plasmids can be grown stably at 30°C, but the plasmid will be lost at 42°C. For removing the plasmid, the strain can be streaken on a antibiotic-free LB agar plate and grown at 42° C overnight. Plasmid loss can be confirmed by streaking the new colonies on a plate with the relevant antibiotic. This feature has been confirmed by Team Uppsala University 2012 for the pSB8C15, in E coli K12 substrains MG1655 (see figure) and DH5α (not shown).

Testing of pSC101ts ori in E coli MG1655. LA plates, A,B are antibiotic-free, C,D with 12 µl/ml chloramphenicol. From left to right on each plate: A. pSB8C15-red and pSB4C15-red grown overnight at 30° C. B. pSB8C15-red and pSB4C15-red grown overnight at 42° C. C. 3 clones each of pSB8C15-red and pSB4C15-red from A. D. 3 clones each of pSB8C15-red, pSB4C15-red grown overnight from B. C and D were grown overnight at 30° C. Due to the low plasmid copy number, red color was not visible until after 48 hours of incubation.

All backbones are available with a pUC origin and a constitutive RFP casette in the BioBrick site. This allows rapid growth at 37° C, high plasmid yields and faster red color expression. Temporary exposure to higher temperatures does not affect plasmid maintenance noticeably, and transformation recovery can be done at 37° C.

Very easy ori replacement is possible in the pSB8x15 backbones. The the plasmid can have the pSC101ts ori cut out with NheI and MluI and another ori ligated in. To remove any religated pSC101ts, the transformants can simply be grown at 42° C. This opens possibilities for much-awaited new orhogonal origins in the BioBrick system.

A problem when doing chromosomal intergration is that some clones may take up a plasmid instead of recombineering it into the chromosome. When doing recombineering with pSB8x15(Frt) backbones, any such clones can be removed by growing them at 42° C. A pSB4x15 backbone with a Flp recombinase gene would of course also make an excellent Flp plasmid.

[1]P. Jensen, K Hammer Appl: "The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters" Environ Microbiol. 64.1 (1998) 82–87.
[2] K.A. Armstrong, R. Acosta, E. Ledner, Y. Machida, M. Pancotto, M. McCormick, H. Ohtsubo, E. Ohtsubo: "A 37×10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1" J. Mol. Biol., 175 (1984), 331–348
[3] T Hashimoto-Gotoh, M Sekiguchi: "Mutations of temperature sensitivity in R plasmid pSC101" J. Bacteriol. 131.2 (1977) 405-412