Difference between revisions of "Part:BBa K1088027:Experience"

Patrick Rosendahl Andreassen

Growth experiments of MG1655 strains carrying either no plasmid (WT), pSB1C3-Pcon-araC-term-Para-HRT2 (BBa_1088016; HRT2), pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B.subtilis) (BBa_1088024; Dxs), or pSB1K3-Pcon-araC-term-Para-HRT2 and pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B.subtilis) (CPS). - and + indicates absence and presence, respectively of 1 mM IPTG and 0.2 % arabinose, respectively as well. The dashed line indicates time of induction with IPTG and/or arabinose. All strains were started at OD600=0.05 from an overnight culture, and were shaked with 180 rpm. A) The HRT2 strain grew slightly slower than the other strains at 37ºC, and upon induction some of the culture even seemed to die, though it catched up with the rest in the stationary phase. B) The CPS strain didn’t reflect what was seen in A when only the HRT2 expression was induced with arabinose. In this experiment WT grew slightly faster than CPS. C) WT and CPS was grown for 3 hours at 37ºC, and along with induction the temperature was lowered to 20ºC. This was done due to a hypothesis about a phenomenon called protein clouding where proteins are overexpressed so much that they misfold and loss their function. By lowering the temperature the risk of misfolding should decrease. All strains grew at the same pace in this experiment.

Patrick Rosendahl Andreassen

We investigated MG1655 strain carrying both BBa_K1088016 on pSB1K3 and BBa_K1088027 (CPS) by phase contrast microscopy and flow cytometry in order to evaluate whether there was a change in size or morphology compared to the wildtype (WT). As indicated by the forward side scatter measurements, there was a difference in size between CPS and MG1655 wildtype both before and after induction in late exponential phase (0.2% arabinose, 1 mM IPTG, 1 mM MgCl2) . Although both strains became small upon induction,the difference in size relation between the CPS and the wildtype remained. The difference in size was less clear during phase contract microscopy.

Patrick Rosendahl Andreassen

Growth curve of dxs devices. Cells were started at OD600=0.005 and incubated at 37oC and 180 rpm. 2 triplicates of MG1655 carrying no vector (WT), empty pSB1C3 vector (pSB1C3), pSB1C3-Plac-dxs(B.subtilis) (BBa_K1088011) (No LacI), pSB1C3-Pcon-lacI-Plac-dxs(B.subtilis) (BBa_K1088027) (LacI(N)), or pSB1C3-Pcon-lacI:LVA-Plac-dxs(B.subtilis) (BBa_K1088013) (LacI(LVA)). At time 2.5 hours one of each triplicate was induced with 1 mM IPTG. No growth change was observed.