Our submitted BioBricks

<groupparts>iGEM015 Berlin</groupparts>

Plasmid Architecture of BBa_K1617002

We successfully constructed 8 BioBricks, which were sequenced and send to the registry. All of our tests were performed using the standard PSB1C3 vector backbone. Functional parts had a strong RBS (BBa_B0034) as well as the J23110 or J23106 Promoter from the Anderson Promoter library. After constructing a working FliC MCS plasmid we inserted engineered D3 domains.

Functionality of our BioBrick Prom110_FliC_MCS (BBa_K1617003)

A = MG1655 z1 ΔFliC, B = MG1655 z1 wt, C = Prom110_FliC_MCS (BBa_K1617003)

To prove and validate that our designed construct works as expected we performed a motility assay. We have designed the flagelline expressing gene FliC with an integrated multiple cloning site and cloned it into PSB1C3 with the constitutive promotor BBa_J23110. The figure shows the comparison regarding the motility between our construct (right) and a negative (left) and positive (middle) control. Since our construct was transformed into E. coli srtain MG1655 z1 ΔFliC we used the highly motile wildtype strain MG1655 z1 as positive control and as negative control the FliC knockout strain MG1655 z1 ΔFliC.

The pictures of negative control and construct were taken after 14 hours of incubation at 37 degrees Celsius. The positive control was taken after 5 hours for means of comparison since after 14 hours of incubation the plate of this control was completely overgrown and therefore not evaluable. Our motility assay reveals that our designed construct which was transformed into a FliC deficient strain is able to successfully express flagellins which are folded correctly and integrated into the surface of the cells. Compared to the highly motile wildtype strain the motility of our construct is less pronounced, nevertheless, in contrast to the negative control there is a significant difference in the motility visible. Our BioBrick enables the FliC deficient strain to be motile again.

Through the Motility Essay it could be shown that our BioBricks work. With cloning the BioBrick into a delta flick deficient strand the motility could be regained. There is no significant difference according the motility between the wildtype and our transformed consturuct in delta flic deficient clones.