Team:Heidelberg/Templates/Indigoidine week22 overview


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T-domain shuffling

Last week's co-transformation experiments showed that exchanging the indC-T-domain with the bpsA T-domain results in a functional indigoidine synthetase, provided specific domain borders were chosen. We now used this domain borders to also try the other native T-Domains T3-9from B. parabrevis, D. acidovorans, P. luminescens and E. coli. We assembled the pRB23 variants T3 to T9 with the domain borders b31 and performed co-transformations with the PPtase plasmids pRB15-18.

We analyzed the data of the spectral absorption measurements versus time and compared the growth synamics and pigment production between different engineered indigoidine synthetases in interplay with different PPTases.

Controlling Indigoidine synthesis

Furthermore we co-transformed E. coli TOP10 with the lacI-Sfp-constructs and pRB22 to determine whether the indigoidine production is controllable.

The lacI-sfp-constructs were not functional because the transformed cells did not turn blue even if induced.
Instead we tried the E. coli NEB Turbo strain which overexpresses the lac repressor. In this way, leaky expression and thereby growth retardation might be prevented.