Team:UPNAvarra Spain/Notebook/Week7
Week 7
12th Agust 2019
We received the sequencing results for mercury and nitrate constructions. The results were different for each one. In case of mercury we could confirm that the sequence was correct, so we concluded our work cloning this sequence. To star working on inductions, we had to streak the culture on agar plates from the glycerol stock, and incubate them at 37ºC.
However, sequencing process gave us bad news for nitrate constructions. In both amilCP and amilGFP containing constructions, the sequence corresponding to the promoter (sensitive to nitrate) was missing. In order to resolve it, we digested these constructions (EcoRI-XbaI) and repeated the ligation of the promoter (EcoRI-SpeI). Then we performed the electroporations.
Regarding lead and arsenic constructions, we performed the minipreps and checked them by digestion. As the colonies seemed to be positive, we sent them to sequencing.
However, sequencing process gave us bad news for nitrate constructions. In both amilCP and amilGFP containing constructions, the sequence corresponding to the promoter (sensitive to nitrate) was missing. In order to resolve it, we digested these constructions (EcoRI-XbaI) and repeated the ligation of the promoter (EcoRI-SpeI). Then we performed the electroporations.
Regarding lead and arsenic constructions, we performed the minipreps and checked them by digestion. As the colonies seemed to be positive, we sent them to sequencing.
13th Agust 2019
We prepared a preinoculum of the mercury construction.
Following with nitrate constructions, we obtained colonies from the electroporation performed yesterday, so we checked them by colony PCR. Showing positive results, we inoculated those colonies on 3 mL of LB medium and left the cultures growing overnight at 37ºC.
Following with nitrate constructions, we obtained colonies from the electroporation performed yesterday, so we checked them by colony PCR. Showing positive results, we inoculated those colonies on 3 mL of LB medium and left the cultures growing overnight at 37ºC.
14th Agust 2019
We performed the induction of the mercury construction using different concentrations of HgCl2. As it was the first attempt, we tested different concentrations and induction times in order to optimize this process.
With regards to nitrate constructions, we performed the minipreps and checked them by digestion. In light of the positive results, we replaced the glycerol stocks and sent these new constructions to sequencing.
With regards to nitrate constructions, we performed the minipreps and checked them by digestion. In light of the positive results, we replaced the glycerol stocks and sent these new constructions to sequencing.