Team:Uppsala University/Backbones/Details

Characterization of copy number is essential when describing a low copy number plasmid. The copy number of the pSB4x15 series, especially the pSB4C15, has been estimated by three methods giving all leading to the same conclusion.

• Measurment of fluorescence by FACS flow cytometry
• Measurment of plasmid yields from liquid cultures
• Visual color development of colonies on plates

Flow cytometry
E coli MG1655 was transformed with plasmids of different backbones with the J04450 standard RFP cassette. Liquid cultures were analysed by fluorescence with a Fluorescence activated cell sorter (FACS), quantitivly measuring the fluorescence of individual cells. Due to the active native lacI repression system in the bacteria, the experiment was performed with and without IPTG for promoter induction. It can be expected that a native lacI system represses a low copy plasmid more than a high copy plasmid. The experiment was conducted very similar to our promoter characterization. A IPTG level of 0.5 mM was chosen to ensure full or nearly full lac induction, as demonstrated by the lacIq repression test (see below).

Quadruplicates (for +IPTG) or triplicates (-IPTG) of E coli MG1655 strains carrying pSB3C5-red, pSB4C5-red, pSB4C15-red, and a DH5alpha strain carrying pSB1C3 with a non-coding construct, were inoculated in 2 mL LB media with chloramphenicol (12 µg/mL) and with or without IPTG (0.5 mM). Cultures were grown shaking at 37° C for 15 hours (+IPTG) or 19 hours (-IPTG), and were visibly confirmed to have grown at steady state for at least 4 hours.

Samples were equilibrated in PBS solution at 1:160 dilution for 2 hours, and then measured by a BD Biosciences FACS. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded.

Results (see figure 1) indicates that that the pEL4C5 has similar or higher copy number than the low to medium copy number plasmid pSB3C5. The pSB4C15 strain shows a lower fluorescence by more than an order of magnitude. In the +IPTG experiment, it expresses less than 1/20 of the flourescence of the other strains.

Plasmid yieds
The cultures of the +IPTG flow cytometry experiment were also plasmid prepped using the Sigma-Aldrich GenElute Miniprep kit, according to the manufacturers instructions. The order of the cultures was randomized during prepping, and elution was done with 100 µL elution buffer.

Significantly higher plasmid yields were observed in pSB3C5 and pSB4C5 than in pSB4C15, suggesting a higher copy number. The variance in yield is however large.

Strong RFP expression was observed in pSB4C5-red strains, weak color in pSB8C15-red strains and no color in pSB4C15-red strains. This points to higher plasmid copy number in pSB4C5 than of the other plasmids. The native lacI system of MG1655 likely repressed the lacI promoter well, inhibiting color development.

Conclusions
Results of fluorescence measurments, plasmid yield and color development on plates all point to the pSB4C5 having a significantly higer copy number than specified, of the same magnitude as pSB3C5. We are confident to conclude that the copy number regulation of pSB4C5 is broken. This conclusion can also be expanded to the other pSB4x5 backbones, since they an identical origin of replication. Casual observations also support this result.

The performed experiments also strongly support that pSB4C15 is a true low copy bockbone. It is strongly suggested that this result is also valid for the whole pSB4x15 series, since they only differ in their resistance cassette.

Repression of common lac promoters
To asses functionality of lac repression and the effect of IPTG induction in the pSB4x15Iq backbones, three common lac-repressed promoters were assembled with red fluorescent protein (RFP) in the pSB4C15 backbone and grown with and without IPTG. Flourescence was measured with a fluorescence activated cell sorter (FACS).

Cassettes of the lacI, LlacO and T5lac and RFP were assembled in the pSB4C15Iq backbone, inE coli DH5alpha. Single clones of these were inoculated in 2 mL LB media with chloramphenicol (12 µg/mL) and with or without IPTG (0.5 mM). Cultures were grown shaking at 37° C for 18 hours, and were visibly confirmed to have grown at steady state for at least 4 hours.

Samples were equilibrated in PBS solution at 1:160 dilution for 1 hour, and then measured by a BD Biosciences FACS. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded.

Figure 4: Relative fluorescence of RFP with different promters in pSB4C5Iq, with and without IPTG induction (0.5 mM). Fluorescence is in arbitrary units.

Fluorescence (arbitrary units) Promoter -IPTG +IPTG pUC-PlacI 0.012 0.97 PLlacO 0.17 4.26 PT5lac 0.06 3.58 PlacI 0.07 1.52 The acquired data clearly shows that the pSB4C5 backbone is capable of tightly repressing promoters on the same plasmid. It also demonstrates that expression can be induced to significant levels by addition of IPTG. Induction at different IPTG levels To asses investigate the IPTG dose response of the lac backbones, a series measurment was also performed on pSB4C15Iq with the pUC-red insert, i e when it is a high copy plasmid. In the same experiment as above, the pUC-red sample was grown at different IPTG concentrations. They were measured as above. Figure 5: Fluorescent dose-response of pSB4C15Iq with different concentrations of IPTG. Fluorescence (arbitrary units) [IPTG] (mM) 0 0.25 0,.5 1.0 The experiment shows that a lacIq system on a high copy plasmid is strongly inhibited by a IPTG concentration over 0.25 mM. Sponsors Retrieved from "http://2012.igem.org/Team:Uppsala_University/Backbones/Details" Recent changes What links here Related changes Special pages My preferences Printable version Permanent link Privacy policy Disclaimers