Team:UPNAvarra Spain/Notebook/Week8
Week 8
19th August 2019
We received the results of samples sent to sequencing. In case of nitrate constructions, we fortunately confirmed that the sequences were correct this time. So, we streak the bacteria from the glycerol stocks on agar plates and left them overnight at 37ºC.
However, we showed that lead and arsenic constructions had important mistakes. The sequence corresponding to the RBS was missing in all of the constructions. Moreover, there were some base-pair substitutions in the sequences corresponding to the promoters. For that reasons, and given the little remaining time to finish our lab work we decided to give up trying to improve these constructions.
On the other hand, we received the DNA fragments from Twist Bioscience and we started working on it. We selected two constructions designed to be cooper and cadmium biosensors, with the amilCP and eforRed chromoproteins as reports in each case, respectively. The first step was the resuspension of the lyophilized DNA products on water. After that we digested these fragments (EcoRI-PstI) and, at the same time, we prepared the plasmid pSB1C3 to be used as backbone for these fragments (EcoRI-PstI digestion). Then, left a ligation overnight at 14ºC.
However, we showed that lead and arsenic constructions had important mistakes. The sequence corresponding to the RBS was missing in all of the constructions. Moreover, there were some base-pair substitutions in the sequences corresponding to the promoters. For that reasons, and given the little remaining time to finish our lab work we decided to give up trying to improve these constructions.
On the other hand, we received the DNA fragments from Twist Bioscience and we started working on it. We selected two constructions designed to be cooper and cadmium biosensors, with the amilCP and eforRed chromoproteins as reports in each case, respectively. The first step was the resuspension of the lyophilized DNA products on water. After that we digested these fragments (EcoRI-PstI) and, at the same time, we prepared the plasmid pSB1C3 to be used as backbone for these fragments (EcoRI-PstI digestion). Then, left a ligation overnight at 14ºC.
20th August 2019
Following with nitrate induction optimization, we prepared a preinoculum (overnight at 37ºC). We also performed the electroporations of the ligation of cooper and cadmium fragments.
21st August 2019
We continued with the induction of the nitrate constructions. We prepared the inoculum and performed an induction with different KNO3- concentrations of both nitrate-amilCP and nitrate-amilGFP constructions. As it was the first attempt, we tested different concentrations and induction times in order to optimize this process. We obtained good results, so we also took some pictures of the pellets to be analyzed during the modeling process.
For the Twist constructions, we performed a colony PCR of the colonies obtained after the ligation. With positive results, we follow the verification of these sequences picking positive colonies on LB liquid media and growing the cultures overnight.
For the Twist constructions, we performed a colony PCR of the colonies obtained after the ligation. With positive results, we follow the verification of these sequences picking positive colonies on LB liquid media and growing the cultures overnight.
22nd August 2019
We spent the day performing some minipreps of final Twist constructions. We digested them with restriction enzymes, and we obtained positive results, with fragments corresponding to the expected sizes for each construction. We also prepared the corresponding glycerol stocks.
In order to be ready for the next week we prepared LB medium in different formats to be used in the following inductions.
In order to be ready for the next week we prepared LB medium in different formats to be used in the following inductions.