Team:UPNAvarra Spain/Notebook/Week6
Week 6
5th August 2019
We followed working with all the constructions, each one in a different point of the process. Regarding mercury, we picked some positive colonies (previously checked by colony PCR) to LB liquid medium and left them growing overnight at 37ºC. In case of lead and arsenic, we checked more colonies by colony PCR from the same plate. We found some positive colonies, with que expected fragment sizes, containing the corresponding chromoproteins. We also inoculated LB liquid medium with those positive colonies and left them growing overnight at 37ºC.
Finally, we started working with the nitrate constructions in order to optimize the induction process with KNO3-. For that purpose, we had to streak each culture on agar plates from the glycerol stocks, and incubate them at 37ºC.
Finally, we started working with the nitrate constructions in order to optimize the induction process with KNO3-. For that purpose, we had to streak each culture on agar plates from the glycerol stocks, and incubate them at 37ºC.
6th August 2019
We performed the minipreps corresponding to the last ligation step of the mercury construction. Fortunately, we found positive results after the digestion with restriction enzyme, so we prepared a glycerol stock and sent them to sequencing.
At the same time, the minipreps of lead and arsenic constructions were performed. We digested them with restriction enzymes and we obtained positive results, with fragments corresponding to the expected sizes for each construction.
For nitrate, we prepared a preinoculum, inoculating 3mL of LB medium with the antibiotic with a single colony from the plate. We left it growing overnight at 37ºC shaking.
At the same time, the minipreps of lead and arsenic constructions were performed. We digested them with restriction enzymes and we obtained positive results, with fragments corresponding to the expected sizes for each construction.
For nitrate, we prepared a preinoculum, inoculating 3mL of LB medium with the antibiotic with a single colony from the plate. We left it growing overnight at 37ºC shaking.
7th August 2019
We performed the last round of BioBrick assembly with the lead and arsenic constructions, in order to add the terminator downstream of the other parts. We digested the previous constructions (EcoRI-SpeI) and the plasmid containing the terminator sequence (EcoRI-XbaI). After that, we performed the ligation and electroporation of both constructions.
We also continued with the induction of the nitrate constructions. We prepared the inoculum and performed an induction with different KNO3- concentrations. We realized that there were no coloring in the cultures after the induction, so we decided to send the constructions to sequencing in order to look for a problem in the sequence.
We also continued with the induction of the nitrate constructions. We prepared the inoculum and performed an induction with different KNO3- concentrations. We realized that there were no coloring in the cultures after the induction, so we decided to send the constructions to sequencing in order to look for a problem in the sequence.
8th August 2019
We obtained a lot of colonies from the ligation of lead and arsenic constructions. We checked some of them by colony PCR, and we observed that in both cases we had colonies harbouring the correct constructions according to the sizes of the PCR products.