Team:Uppsala University/Backbones

There have been longstanding doubts about the behaviour of the existing pSB4x5 series of low copy plasmids. We can now demonstrate, with fluoresence measurements by flow cytometry, that the old low copy plasmids has a considerable higher copy number than specified.

Due to a need for a low copy BioBrick plasmid in our project, we have thus devoloped a new series of BioBrick standard vectors. The new pSB4x15 backbones have a low copy pSC101 replication origin (~5 copies per cell) and ampicillin, chloramphenicol, kanamycin or spectinomycin antibiotic resistance markers. They are especially designed 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.

Team Uppsala University recommends the pSB4c15 series as a replacement to pSB4c5 for all low copy plasmid applications.

The pSB4x15 series in brief:

• Verified low copy number
• More accurate annotation
• No homologies to the E coli genome that could interfere with Lambda red recombineering
• Easy introduction of new resistance cassettes
• Easy introduction of new orthogonal replication origins
• Smaller backbone size
• Flp recombinase target sites around the resistance cassette available
• LacIq versions for tight repression available
• Thermosensitive versions 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, regardless of resistance cassette or insert in the cloning site. 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 flanked by NheI and MluI restriction sites for easy switching of origin of replication, preferably replacing the thermosensitive pSB8x15. An illegal SpeI restiction site has been removed from the origin.

Characterization

Figure 2: Relative fluorescence of red cassette (J04450) in different backbones in E coli MG166, with and without IPTG induction (0.5 mM). Quadruplicates (+IPTG samples) or triplicates (-IPTG). Fluorescence in arbitrary units, not compareable between +IPTG and -IPTG. Methods.

The copy number of pSB4C15, as compared to pSB3C5 and pSB4C5, has been estimated by flow cytometer fluorescence (see figure 2) and plasmid prep concentration measurements. According to our measurements, our pSB4C15 is present at a stable copy number that is a fraction of that of the other tested plasmids. pSB3C5 and pSB4C5 have a similar copy number, with pSB4C5 slightly higher. This is also consistent with the experience of other teams [1, 2]. Read details and methods.

The classic pSB4C5, and most likely the whole pSB4x5 series, are not low copy backbones as specified in the registry. They should not be used as low copy backbones. A possible future use of the pSB4x5 series is as a middle copy backbone that is compatible with the existing pSB3x5 (with p15A ori), something that is certainly useful from a syntetic biology standpoint.

Nomenclature

• pUCori - a high copy origin of replication. Amplified from the pSB1A3 backbone.
• red - the standard PlacI-RFP cassette J04450.
• redq - a RFP cassette 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.

All pSB4x15 backbones have with a pUC origin and the standard RFP cassette in the BioBrick site. This facilitates high plasmid yields and fast red color expression.

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 cassette on the plasmid ensures that the copy number of the lacIq gene always follows that of your inserted genes, providing guranteed strong repression without inducing unneccessary metabolic load.

Repression and induction has been shown to be functional by fluorescent measurements of the lacI, lacIq, T5lac and LlacO promoters expressing red fluorescent protein in pSB5C15Iq. Read details and methods.

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 streaked 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 6) and DH5α (not shown).

Figure 6: 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 lacIq backbones are available with a pUC origin and a constitutive RFP cassette in the BioBrick site. This allows rapid growth at 37° C, high plasmid yields and faster red color expression. In our expreience, temporary exposure to higher temperatures does not affect plasmid maintenance noticeably, and transformation recovery can be done at 37° C.

Origin switching
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 orthogonal origins in the BioBrick system.

Reliable recombineering
A problem when doing chromosomal intergration is that some clones may take up the template plasmid instead of recombineering the PCR product into the chromosome. When doing recombineering with pSB8x15(Frt) backbones, any such clones can be removed by growing them at 42° C. A pSB8x15 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