Team:Uppsala University/Backbones/Details

The copy number of the pSB4x15 series, especially the pSB4C15, has been estimated by three methods all pointing to it being maintained at a consistent low copy number in E coli cells.

• 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.

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 in a BD Biosciences FACSaria III. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded.

Results indicate that that the pSB4C5 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, and even is lower without induction.

Plasmid yields
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. Concentrations were measured with a Nanodrop 1000 spectrofotometer. All measurements yielded good curves.

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.

Color development
E coli MG1655 carrying pSB4C5-red, pSB4C15 and pSBC15 was plated on on LA plates with chloramphenicol (12 µl/mL) and incubated at 30° C or 37° C for two days. Color expression was assed visually.

Figure 3: Color assesment plates grown for two days. In clockwise order: A. Three clones each of pSB4C15-red and pSB4C5-red. Grown at 37° C. B. Two clones each of pSB4C15-red, pSB8C15-red and pSB4C5-red. Grown at 30° C.

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.

Conclusion
Results of fluorescence measurments, plasmid yield and color development on plates all point to pSB4C15 being a true low copy backbone. It is strongly suggested that this result is also valid for the whole pSB4x15 series, since they only differ in their resistance cassette.

The results also show that pSB4C5 has 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.

Thus, we recommend replacing the pSB4x5 series with the pSB4x15 for all low copy applications

IPTG induction
To investigate the functionality of lacIq 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 Plac, PLlacO (BBa_R0011) and PT5lac (BBa_K592008) promoters and the pUCori-Plac were assembled with RFP in the pSB4C15Iq backbone, and transformed into E 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 in a BD Biosciences FACSaria III . 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Since the purpose of the experiment was to investigate overall repression functionality and the possibility of induction, the samples of with Plac, PT5lac and PLlacO can be considered a triplicate measurement.

Conclusions
Results of fluorescence measurments, plasmid yield and color development on plates all point to pSB4C15 being a true low copy backbone. It is strongly suggested that this result is also valid for the pSB4x15Iq series, since they share an identical origin. The IPTG repression data demonstrates that the pSB4C5Iq backbone is capable of tightly repressing promoters on the same plasmid, whether it is present in high or low copy number. 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.