July, 28th

Production Team

Paola Gallardo. We run an electrophoresis to check the result and we observed a lot of unspecific very intense bands, but the expected bands had few intensity. We isolated and purified the desired bands. We digested the products: gltD with XbaI and PstI, fecI-fecR with EcoRI and PstI for two hours. We joined them with different vectors and rested o/n.

David Caballero. fecA* inocula appeared in red color behind being incubating all night long. This meant that bacteria had plasmids with RFP (red fluorescent protein) instead of fecA*. So we could not use it. Checking fecA* plates we saw that colonies which come from our inocula were red two days after start incubating. We found a new white colony too, so we analyzed it by colony PCR and electrophoresis analysis, but the result was negative. Changing focus, we analyzed gltB and fecI-fecR* inocula: vector purification, restriction enzyme parts digestion and 0.8% agarose gel electrophoresis analysis. We had a positive result for gltB part and negative for fecI-fecR* part (not well digested). With this it had been tested a new synthesized part: gltB. The loss of fecI-fecR* is unimportant, since it had already been gained in another process made by a partner. By this moment, we had got all searched parts by PCR saving fecA*. It was resisting.

Assembly Team

We have to test the UPO 1+2 candidate so we digest with Nhe1 to UPO1 and we can see the plasmid run into the gel like a lineal plasmid. Thus, finally we can say we have UPO 1+2.

And again, we start with UPO 1+19 and UPO 12+2, 12+19, 13+3 y 1+2+16+3.