Team:Hamburg/Composite Part

Best Composite Part: BBa_K2588021

To create a Sustainable H.I.E.L.D. we needed the bacteria not to grow and divide, but to maintain a stable cell density. Our growth inhibition module BBa_K2588021 was confirmed by characterization. It inhibits E. coli cell division and growth to a third of its natural growth within only 6 hours after induction. BBa_K2588021 induces expression of growth inhibiting proteins cbtA, cspD and mraZ in absence of diaminopimelic acid (DAP). This allows to grow bacteria wearing this construct by addition of DAP but becomes active in natural environments without high amounts of DAP (Fig. 1).

Fig. 1: BBa_K2588021 Map. DAP-repressed promoter dapAP induces expression of cell division inhibitors cbtA, cspD, and mraZ.

To characterize our growth inhibition module, we measured the optical density of the inhibited (+DAP), induced (-DAP), positive (DH5α) cultures at OD600 nm for 6 hours at 1-hour intervals. The part was assembled using the Golden Gate assembly method. The growth inhibition module is regulated by a dapAP promoter, which is inhibited by the presence of diaminopimelic acid. Measurement is shown in Fig. 2.  The culture with (-DAP) had a reduced growth by factor 3.21 compared to the control. The culture with 40 µM DAP added had a reduced growth by factor 2.30.  

Fig. 2: Characterization of BBa_K2588021 Cultures containing the growth inhibition module were inhibit with DAP and OD600 was measured for 7 h in 1 h intervals. Uninhibited cultures as well as DH5α were measured as positive controls.

Upon addition of DAP (+DAP), cultures containing the growth inhibition module showed a growth comparable to the positive control, while the induced growth inhibited culture (-DAP) exhibited weak growth. While we expected the induced growth inhibited culture to not proliferate and not growth, a increase in growth rate was observable. Therefore we confirm that the growth inhibition module indeed reduces cell proliferation.

Funding