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.

Chassis

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.