Difference between revisions of "Part:BBa K1073034:Experience"

iGEM Team Braunschweig 2013

Using ampicillin containing complex medium, growth of cells containing this device could be induced by adding synthetic autoinducer N-3-oxododecanoyl homoserine lactone to the culture broth. The required transcription regulator LasR is encoded in the device. Cells also exhibit a dark blue color due to the constitutively expressed aeBlue chromoprotein. Thus aeBlue was used as a selection marker identifying cells containing this device. 2xYT medium containing ampicillin was used for cultivation and cells were incubated at 37°C and 250 rpm in non-baffled flasks.

iGEM Team Braunschweig 2013: Comparison of growth curves of E. coli JM109 containing BBa_K1073034 on the high copy plasmid pSB1C3 in the presence and absence of autoinducer N-3-oxododecanoyl-HSL in the culture broth. Clavulanic acid was added to eliminate background activity of beta lactamase due to the leakiness of the promoter.

Due to the leakiness of the las promoter the beta lactamase gene ampR is also expressed to some extend when no N-3-oxododecanoyl-HSL is added to the culture broth. Therefore, a low concentration of clavulanic acid (5 µM) was added to the broth which acts as beta lactamase inhibitor and eliminates background activity of the beta lactamase.

Growth of cells could also be induced when culture supernatant of BBa_K1073035 containing N-3-oxododecanoyl synthesized by LasI encoded in BBa_K1073035 is added to cultures on agar plates containing ampicillin.

iGEM Team Braunschweig 2013: Growth of E. coli JM109 containing BBa_K1073034 on the high copy plasmid pSB1C3 induced by the supernatant of expressed BBa_K1073035. The supernatant contains N-3-oxododecanoyl-HSL produced by LasI. A piece of filter paper was soaked with supernatant of a BBa_K1073035 and placed on the agar plate containing ampicillin (0.29 mM). Cells are only growing close to the filter paper.