Suicide System

We received the sequencing results of RBS_B0031-Tse2-TT_B0015 construct and double checked before the BB submission. At this point we were able to clone it downstream the T5LacOperator Promoter (BBa_K875002) to test its functionality. We screened the colonies and two of them resulted positive (T5LacOp-B0031-Tse2-B0015), so we plated them with and without IPTG. One of the two colonies doesn't grow on IPTG and grew without it! We also did the same test in LB culture and the same colony died with IPTG and grew without it.

We also recorded the OD of the LB culture IPTG induced and not induced at fixed time intervals for several hours during the day. We designed the growth curve.

Antibody

To get a proof of pelB-scFv and LPP-OmpA-scFv expression, we used standard techniques as SDS-PAGE and Western blotting. The results showed that in both cases scFv is expressed. LPP-OmpA is expressed in great quantities. PelB-scFv is present only in traces. We have suspects that pelB-Ab is toxic for this E. coli strain.

Cumate-Switch Regulation

CymR
We cut the double CymR Eco/Pst and we ligated it in the plasmid for the integration using K510012 E/P. So we transformed in the C118 cells and we obtained few positive clones. We extracted the plasmid from the positive clone and we co-transformed it with the plasmid containing the transposase pTNS2 in both DH5alpha and Nissle cells. We made two cotransformation:
- 0.5ng of pTNS2 and 0.5ng of K51002-CymRx2
- 1ng of pTNS2 and 1ng of K51002-CymRx2
We made a colony PCR with specific primer and we obtained some clones that had the double CymR integrated. Unfortunately the sequence of this construct was wrong because the J23100 wasn't in the plasmid.

T5 PROMOTER - CUMATE OPERATOR
We digested again the T5CuOperator-pSB1C3 S/P and the E0240 X/P in order to ligate them together. We find some positive clones and we analyze them with an E/P. The cut was correct. So we eluted the fragment T5-operator-E0240 X/P to ligate it in the plasmid J61002 containing J23100-CymR-B0015. We didn't find positive clones.