Team:Heidelberg/Templates/Indigoidine week18 overview


T-Domain Shuffling

We again assembled pRB19 from pRB14 and did all T-domain-exchanges in this plasmid. After we picked and prepped positive colonies we performed co-transformations with pRB15-18 to see which PPTases are able to activate any of the T-domains inserted into indC.
To obtain experimental validation that the strategy to use two separate plasmids works and to verify that the PPTase plasmids are free of template backbone we co-transformed TOP10 cells with pRB21 and pRB15-18.
In parallel we assembled pKH4, which is derived from pRB14. The sfp coding sequence was replaced by a nonsense part of pMM65 using the cutting sites BamHI and NheI. In this way, we were independent from the success of the assembly of pRB19.

BioBricks for the Registry

After the corrected primer RB78 arrived we assembled pRB22 and cloned the BioBricks pKH6-10 for the part submission.