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From 2013.igem.org

T-Domain Shuffling

We started inserting new T-Domains into indC to check their functionality. In total we first assembled 19 variants of pRB19, which are named pRB19-Txx. The appended three-letter code identifies the respective T-domain or TTE-domain and is listed on our plasmid page in the materials section.

Since PCR screenings of pRB19 were inconsistent, we started with replacing the T-Domain of pRB14, which contains sfp as a PPTase. Screenings and sequencing results showed that assembly and transformation were correct but all colonies except for that on the positive control retained a white phenotype. We tested all the other T-domains on the newly assembled pRB19 with every PPTase pRB15-18 next week.

T-Domain Shuffling – PPTase Plasmids

As the PPTase plasmids pRB15-18 showed red colonies after retransformation of a positively prepped colony, we picked new colonies and prepped them. We will do the co-transformation experiments with the new version of pRB15 but with the old ones of pRB16-18.

BioBricks for the Registry

This week we removed the two internal RFC[10]-cutting sites of indC (EcoRI and SpeI) on pRB21 using a CPEC approach to yield pRB22]. The modified indC (indC*) was amplified introducing the RFC[10] prefix and suffix (Primer KH21/22). The four PPTases and indC* coding sequences were cloned into pSB1C3 using standard BioBrick Assembly.

Due to faulty primer design of RB71, the resulting plasmid pRB22 was incorrect as well. We ordered a corrected version of RB78 and assembled the plasmids again in the following week.