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Team:USyd-Australia/pUS201
From 2014.igem.org
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pUS201 Low Copy Plasmid
Aim
To produce a low copy plasmid backbone for the cloning of integron integrase (IntI1). The integron integrase (IntI1) is believed to be toxic to cells when expressed at high concentrations, thus, it was expected that it would benefit the stability of the integrase construct if it was both controllable, and expressed on a low copy plasmid backbone.
Back to topApproach
We sought to improve one existing low-moderate copy backbone, pSB6A1, for this purpose. pSB6A1 is derived from pBR332, having a pMB1 ori site, a Rop gene that acts to reduce copy number and ampicillin resistance as the selectable marker. Additionally, pSB6A1 has had a biobrick prefix/suffix cloning site suffix cloning region added, which has disrupted the tetracycline resistance gene present in pBR332.A large region of defective TetR gene sequence is still remaining, and the cloning region lacks terminators either side of the prefix and suffix. Thus, we sought to assemble a new plasmid, pUS201, incorporating the AmpR, Ori and rop sites from pSB6A1, and the biobrick prefix/suffix cloning region with terminators from pSB1C3.
Figure 1 – General strategy behind the construction of pUS201.
Materials
- DNA
- BBa_J04450: Used to PCR out the pSB6A1 backbone
- BBa_K59009 : Used a source of the BioBrick cloning region
- Primers
Method
PCR of the pSB6A1 backbone (from pSB6A1 containing BBa_J04450 was done using primers 1401 and 1402. Similarly, PCR of pSB1C3 (from pSB1C3 containing BBa_K59009 yielded the biobrick cloning region. The overlap between the sequences of the PCR products allowed for Gibson assembly of the final plasmid, pUS201. This was transformed into Top10 E. coli, with the correct clone selected for on ampicillin, with a white colouration.
Figure 2 – Digestion of the plasmid preparation of pUS201 with a variety of restriction endonucleases yields the expected fragment sizes when visualised with agarose gel electrophoresis. The results are as expected providing us with good evidence that the part is what we want.
Back to topValidation
We aimed to validate that pUS201 was low copy by observing the proliferation of pUS201 positive E. coli in different concentrations of ampicillin or carbenicillin. Unfortunately, the results indicated that pUS201 was not low copy, with the rate of proliferation of bacteria in solutions of LB and carbenicillin similar to pUC19, a high copy plasmid that expresses ampicillin resistance.
Figure 3 – Proliferation of E. coli after 2.5 hours in LB media with various concentrations of carbenicillin, as measured by OD600. Error bars represent the standard error of the mean. pUS201 seems to behave similarly to pUC19, the high copy plasmid, as opposed to the low copy pSB6A1.
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