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Team:TU Darmstadt/Results/Safety
From 2014.igem.org
Safety
Despite all efforts we were not able to assemble the planned test plasmid. The first assembly step was cloned and verified succesfully. The second assembly step in which the T3-RNA-polymerase (BBa_K346000) was supposed to be cloned into the verfied construct however was not successful. Even after multiple attempts we were not able to integrate the Biobrick into our construct and complete neither the test plasmid nor the self-killing plasmid.
In order to still be able to characterize the functionality of the hokD-BioBrick (BBa_K149708) we cloned it downstream of the araC-Pbad-promoter (BBA_K808000) including a ribosome binding site and transformed the construct into E. coli (TOP10). Successfull transformed cells were selected via double selection with chloramphenicol (CMP) and glucose.
Positive transformants were incubated over night in LB-media containing CMP and glucose (2g/l). The overnight culture was then splitted equally into two liquid cultures with LB media and CMP. One culture contained 2 g/l glucose and the other one contained 0.3 g/l (2 mM) arabinose. E. coli containing pSB1C3 were incubated in LB media with CMP as a control culture. After 48 hours of incubation (37°C, 120 rpm) the different cultures were diluted several times and spread on LB-agar plates containing CMP in order to determine the amount of living cells (CFU/ml).
Fig.1: LB-CMP-plates from the spread-plate assay after 24 hours of incubation. Left: control culture; Middle: culture grown with 2 g/l glucose; Right: culture grown with 0.3 g/l arabinose
Addtionally to the spread-plate assay a 105-fold dilution of each culture was plated on LB-CMP-agar plates containing either 2 g/l glucose or 0.3 g/l arabinose and incubated for 24 hours at 37°C in order to investigate the ongoing proliferation of the bacteria with glucose and arabinose.The amount of colonies formed by the control cultures were identical for both types of plates (136 CFU with glucose, 132 CFU with arabinose).
Fig.2: calculated CFU per ml culture for the three different cultures
The culture containing the hokD-gene cultivated in LB-media with glucose showed simmilar amounts of colonies on the plate containing glucose as the control culture (134 CFU) but a 2-fold reduction in CFUs on the plates containing arabinose (63 CFUs). The culture containing the hokD-gene which already had reduced cell-growth because it was cultivated in LB-media containing arabinose only formed 3 colonies on the plate containing glucose and no colonies on the plate with arabinose (Fig.3).
Fig.3: counted number of colonies for the three different cultures on plates containing glucose or arabinose
These results indicate that the expression of hokD leads to a reduction of cell proliferation. The bacteria cultivated with glucose showed no significant reduction in growth rate until they received arabinose which means that the expression of hokD can be controlled by the araC-PBad-Promoter without having a negative influence on cell growth under laboratory conditions. Therefore our designed hokD-BioBrick shows potential to be used in a containment approach for E. coli.
