Part:BBa_C0012:Experience

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UNIQ26aba64b35c20fa7-partinfo-00000000-QINU UNIQ26aba64b35c20fa7-partinfo-00000001-QINU

••••• P.R.A

Prove that natural LacI (BBa_K1088018) has improved function FACS results and growth curves of lacI(N) and lacI:LVA carrying strains. Two triplicates of MG1655 strains carrying either pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B. subtilits)-GFP (BBa_K1088026) (lacI(N)) or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs(B. subtilits)-GFP (BBa_K1088009) (lacI:LVA) were grown from OD600 0.005 to approximately 0.2. At this OD the one triplicate of each strain were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, 150 and 180 min. A) lacI(lva) and lacI(N) strains grew at same pace both with and without induction. B) Per cent of population above fluorescence threshold. Both lacI(lva) and lacI(N) represses the expression, when not induced with IPTG. lacI(lva) reaches a maximum of merely 70-75 % after 150 min, which is both a poor response time and maximum cell per cent fluorescent compared to lacI(N). lacI(N) reaches a maximum just below 100 % at a time between 30 and 60 min. C) Mean GFP fluorescence of entire population. These results reflect what is seen in B, and clearly indicates that overexpression of natural LacI instead of LacI:LVA is the better, when a quick response and high level of expression is wanted.

••• P.R.A

FACS results and growth curves of +/-lacI:LVA carrying strains. One triplicate of MG1655 (WT) and two triplicates of MG1655 strains carrying either BBa_K1088008 (-lacI:LVA) or BBa_K1088009 (+lacI:LVA) were grown from OD600 0.005 to approximately 0.2. At this OD the MG1655 triplicate and one triplicate of each strain carrying constructs were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, and 150 min. A) Growth curve shows that WT grows slightly faster than strains bearing plasmids. B) Per cent of population above fluorescence threshold. None of the WT cells were fluorescent, almost all of the -lacI:LVA cells were constitutively fluorescent, and only cells overexpressing LacI:LVA weren’t fluorescent when not induced. Upon induction increasingly per cent of +lacI:LVA became fluorescent and reaches a maximum of 70-75 per cent after 90 min. C) Mean GFP fluorescence of entire population. The -lacI:LVA cells became increasingly more fluorescent over time, both with and without induction. Though, the induced cells were slightly more fluorescent, which is probably because of the relief of repression from LacI naturally present in the cells. For the +lacI:LVA cells the results from B is reflected.

Jasonk 15:15, 26 July 2006 (EDT):The LVA degradation tag leads to rapid degradation and as a result seeing significant repression with C0012 is unlikely. BBa_C0013 is a lacI with no LVA tail (though at the moment it is not constructed).