Suicide System

We transformed also the double terminator (BBa_B0015) as Bglbrick. We amplified by PCR the BBa_B0015 miniprep with the appropriate PCR protocol. We loaded the gel with the amplified and the eluted was ligated in the pGEM vector.

The plasmid was amplified with the primers SP6 and T7 and then cut BglII/XhoI.

For the final construct -holin LL 37- : the LL 37 (BBa_K875009) was cloned downstream the T5CumateOperator (BBa_K875001), heat inactivated and dephosphorylated at the extremities.

Antibody

We developed an E.L.I.S.A. test to check out if LPP-OmpA-scFv is displayed on bacterial surface. We tried to immobilize the bacteria expressing our antibody on E.L.I.S.A. plates. This experiment had no success probably because it was not optimized enough.

Chassis

CymR
We tryed to insert the double CymR in the K510012 plasmid but it didn't seem to work and we also obtained some strange cuts. Moreover from the sequencing we knew that we had no double CymR but only a single CymR. So for the lack of time we decided to try only the single CymR integration.
T5 PROMOTER - CUMATE OPERATOR
We sequenced the clones and we found that they were right.

We did two different Cumate tests:
In the plate assey

We streak the right clone containing the J23100-CymR-B0015-T5CumateOperator-I13504 in LB agar plate with different concentration of Cumate;

In liquid assay:

We inoculated the right clone containing the J23100-CymR-B0015-T5CumateOperator-I13504 in 20 ml of LB over night. The day after we diluted it untill the OD600=0.2 and when the culture reached OD600=0.6 we induced this colture with 0; 5; 15; 20; 30; 60; 120mM of Cumate