Suicide System

Due to the negative results of the previous week , we decided to use another toxin from the registry, that we have already extracted from iGEM kit and amplified it through the transformation of DH5-alpha competent cells: Tse2 toxin (BB_K314200).
We began to create our toxin construct, cutting Tse2 Toxin (EcoRI/SpeI) to clone it inside the double terminator BB_B0015, previously cut with EcoRI/XbaI.
Unfortunately we did not obtain a positive colony, so we tried different cloning approaches.

Antibody

We replaced pelB leader sequence with LPP-OmpA into the SIP fragment in order to attach the SIP on the bacterial surface. Then we cloned it downstream TLacO promoter. This construct was also tested in W3110 with p-REP 4. We induced its expression with IPTG (1mM). Following the same protocol, and we analysed the induced culture by Western blotting. Our fusion protein is being expressed.

Cumate-Switch Regulation

CymR
We tried to transform again the same ligation and this time we get two positive clones. First we made also a control-cut with EcoRI to exclude errors in the plasmid sequence. Then we cloned the CymR - B0015 fragment in the plasmid J61002 downstream the J23100 promoter. We got no positive clones with the colony pcr so we tried two different kind of ligation:
- the same as before but with the addition of SAP (shrimp-alkaline phosphatase) on the J61002 - J23100 after the digestion.
- a ligation without gel purification and then we selected the non-red colonies.
At the end we obtained some positives from the first procedure.

T5 PROMOTER - CUMATE OPERATOR
We cloned in the two positive clones the E0240 (GFP) downstream the T5 promoter - Cumate operator. After the transformation we obtain no positive clones so we tried to repeat the same process just adding the SAP after the SpeI/Pst cut.
We tried also to make a ligation without purification but with no positive results.
We cut the positive pcr clones with XbaI/PstI but analyzing them in the gel we saw three different bands in every single clone that we cannot identify. So we re-digested other positive clones.