Part:BBa_K864017:Experience

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Team Uppsala University iGEM 2012 The plasmid loss function has been tested by iGEM Uppsala 2012. Our result indicates reliable plasmid loss after growth at 42° C. Color development when carrying a RFP cassette suggests the plasmid copy number is low, but higher than the low copy pSB4C15 vector. Plasmid loss LA plates, A, B are antibiotic-free, C, D with chloramphenicol. From left to right on each plate: A. pSB8C15 and pSB4C15 grown overnight at 30° C. B. pSB8C15 and pSB4C15 grown overnight at 42° C. C. 3 clones each of pSB8C15 and pSB4C15 from A. D. 3 clones each of pSB8C15, pSB4C15 grown overnight from B. Due to the low plasmid copy number, red color was not visible until after 48 hours of incubation. pSB4C15 and pSB8C15, carrying the BBa_I52002 RFP cassette, were transformed into E coli MG1655. Cells with each plasmid were streaken identically on a pair of antibiotic-free LA plates and grown at 30° or 42° degrees overnight. Three colonies of each strain from each temperature were picked and streaken on LA plates with chloramphenicol (12 µg/ml), which were grown at 30° C overnight. All pSB4C5 clones from both plates grew. The pSB8 clones from the 30° C plate grew, but not the clones from the 42° C plate (see figure). The experiment was also performed with E coli cloning strain DH5alpha, with identical results (not shown). This indicates that the pSB8C15 plasmid is lost by growing at 42° C. Copy number E coli MG1655 carrying different backbones with RFP cassette grown for two days at 30° C. Two clones of each, clockwise: pSB4C15, pSB8C15 and pSB4C5. The copy number of the pSB8C15 has not been measured by accurate quantitive methods by us. One could assume it would be similar to that of pSB4C15, due to the very similar ori. However, experience with for example pSB4C5 shows that small changes in an origin could greatly affect the plasmid copy number. As a simple estimation, two clones each of E coli MG1655 with the medium copy plasmid pSB4C5 or the low copy plasmid pSB4C15 were plated together with pSB8C15, all carrying the BBa_I52002 RFP cassette. The plate was incubated at 30° C for two days and visually inspected for RFP expression. Both pSB8C15 clones expressed a weak red color, while the pSB4C5 clones were strongly red and the pSB4C15 did not show any visible color. Similar patterns can be seen on the plasmid loss testing plates above. This result suggests that the plasmid copy number of pSB8C15 lies between that of those two other backbones.

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