Team:Heidelberg/Templates/Indigoidine week20 overview



T-Domain Shuffling

We prepared pRB23-T1 to -TTE19 and performed transformations in E. coli TOP10 as well as co-transformations with the four PPTase plasmids pRB15-18 and a pSB3K3-plasmid with a nonsense insert. This shows whether there is a dependency of T-Domain activation on the PPTases and if there are differences in indigoidine yield using different combinations of T-Domains and PPTases. The co-transformation with a pSB3K3 plasmid with a nonsense insert shows whether the smaller size of the colonies in our co-transformation experiments is due to the co-transformation itself or to the PPTases.

We sequenced every variant of pRB23-Txx and they were all correct. The cotransformation experiment showed, that some of the engineered indigoidine synthetases are still capable of producing the blue pigment. The indigoidine production strongly differs among the various T-Domains. All the combinations are shown in a big table in the detailled lab journal below.

T-Domain Borders

This week we tried to assemble pRB23-b11 to -b34, which are 12 pRB22-derived plasmids, in which the indC-T-Domain was exchanged by the bpsA T-domain. In every variant, the domain borders were defined differently. Those constructs were co-transformed with the four PPTase plasmids pRB15-18 as well as with a pSB3K3 plasmid containing a nonsense insert (Transformation strain: E. coli TOP10). The appearance of blue colonies on the plates showed which domain borders work best for the domain exchange between functionally related NRPS.

The cotransformations of the pRB23-bxx CPEC assembly products with the PPTase plasmids was not successful. Therefore, we prepared plasmid DNA of the pRB23-bxx variants and repeated the co-transformation experiments.

T-Domain Shuffling – PPTase Plasmids

As there is always leaky expression of indC* - due to the fact that E. coli TOP10 lacks the lac repressor and we use a lacPromoter - we assembled variants of the PPTase plasmids which also carry the lacI gene with a lacPromoter.

Engineered indC - Quantitative Assay

Since we want to characterize the indC*-variants and PPTases in a quantitative manner, we establish an assay for measuring the correlations between T-domains, PPTases, growth rate and indigoidine production using OD measurements. We take 96 well plates and measure absorption spectra of various indigoidine producing cultures.