Part:BBa_I50042:Experience

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Applications of BBa_I50042

• As a middle copy origin, compatible with the more common p15A in the pSB3x5 series
• BioBrick standard vector construction

User Reviews

UNIQ4dce159f696004bd-partinfo-00000000-QINU

• User:agynna

iGEM Team Uppsala University 2012 Warning: This is not a low copy origin. The copy number of the pSB4C5 plasmid, with a I50042 ori, has been estimated by three methods and compared to the pSB3C5 (with p15A ori) and the pSB4C15. The results shows strong evidence of pSB4C5 having a significantly higher copy number than specified, comparable to or higher than pSB3C5. Measurement of fluorescence by FACS flow cytometry Measurement 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. Relative fluorescence of red cassette (J04450) in different backbones in E coli MG1655, with and without IPTG induction (0.5 mM). Quadruplicates (+IPTG samples) or triplicates (-IPTG). Fluorescence in arbitrary units, not compareable between +IPTG and -IPTG. Error bars are standard deviation. Samples were equilibrated in PBS solution at 1:160 dilution for 2 hours, and then measured by a BD Biosciences FACSaria II. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Fluorescence (arbitrary units) Plasmid -IPTG +IPTG pSB3C5-red 24.90 29.68 pSB4C5-red 34.53 30.07 pSB4C15-red 0.19 1.34 Control - 0.01 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. 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. Concentrations were measured with a Nanodrop 1000 spectrofotometer. All measurements yielded good curves. Plasmid prep yields. Error bars are standard deviation. Plasmid Average yield pSB3C5-red 114 pSB4C5-red 90 pSB4C15-red 28 - 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 on plates 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. 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. Conclusions The results also demonstrates 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 all share the I50042 origin of replication. Casual observations also support this result. 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.

••••• Reshma Shetty

BBa_I50042 is a functional replication origin. Based on typical miniprep yields of plasmids with BBa_I50042, it appears to be a low copy plasmid. However, the plasmid copy number has not been verified.

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