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Team:NRP-UEA-Norwich/Week2
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Contents |
DESCRIPTION OF WEEK TWO TO BE UPLOADED AT A LATER DATE
While two members of the team worked in the labs on the transformation of Escherichia coli the rest of the team continued with the research into previous BioBricks and those that we felt the characterisation of would be useful to our project. As one of our proposed ideas for the project is to produce a BioBrick that can sense nitric oxide and then go on to increase nitric oxide amounts to toxic levels if appropriate as a potential cancer therapeutic we decided to look into genes for nitric oxide synthase and nitrite reductase, both enzymes that result in the production of nitric oxide. We also looked into plasmids and genes that may be used in mammalian cells as well as bacterial in order to start thinking about where our project could go once the BioBrick is synthesised.
Transformation of the three BioBricks was redone. Two controls were also set up in order to point us in the right direction with what may have gone wrong; the positive control used BRAF (BL21 pLysS) cells which contain vectors for chloramphenicol resistance; the negative control used the NEB 5-alpha cells used in the previous experiment; our aim was for the positive control to show whether the issue lies within the chloramphenicol resistance, or whether the problem lies within the competent cells themselves.
A sample of lysogeny broth (LB) as also inoculated with pre-made culture containing the BL21 pLysS cells in preparation for the next day's plasmid isolation focusing on chloraphenicol resistance.
Protocols for the experiments can be found Here
As we are looking at producing a potential cancer therapeutic as part of our project we focused on looking at how that particular application could be used. Our main idea is to attach a nitric oxide-sensing promoter to an enzyme producing more nitric oxide (NO) in order to bring NO levels to a toxic threshold. We have found two key classes of NO-producing enzyme that may be applicable to our project: the first is nitric oxide synthase (NOS), most commonly from Nocardia species (known as NOSNoc) as it is a bacterial form of NOS, however so far we found little information on the intricacies of the enzyme and therefore feel it may not be the best enzyme to use; the other enzyme we have looked at is nitrite reductase (NiR), specifically copper-based NiR as it has a much cleaner reaction mechanism (producing just NO and H+) compared with other species of NiR.
Rebecca and Lukas went back into the labs today in order to try and transform the E. coli once again. Using the knowledge we had gained through research into why the previous transformations had been unsuccessful agar was used that had a reduced amount of chloramphenicol (50µg/ml to 25µg/ml) in order to try and grow more cells.
We were also visited by Josh Wright from Bioline who very kindly donated a large amount of plasmid isolation kits and PCR gel clean-up kits, as well as giving us a discount on Bioline products. This kind of support from Bioline is extremely important to our project and we are very thankful!
Following Josh's visit Lukas, Russell and Khadija decided to try out the plasmid isolation kits for the first time as they tried to extract the chloramphenicol-resistance plasmid from some cultured E. coli. The kits were extremely easy to use, however in keeping with our previous problems with growing E. coli we were unable to extract the plasmid, which was proven to us by a blank agarose gel picture.
The protocol for all lab work can be found Here
Following the blank agarose gel image more E.coli was inoculated into culture in hope that the lack of DNA was due to an insufficiency in the number of cells.
A/B: Cells with Bba_K381001 + pSB1C3 plasmid (20µl and 200µl)
C: Cells with Bba_K325100 + pSB1C3 plasmid (200µl)
D: Cells with Bba_K325909 + pSB1C3 plasmid (small colonies)
E/F/G: Cells with Bba_K381001 + pSB1C3 plasmid(200µl)
Positive control: colony of Cells with plys plasmid (Chlorophenicol resistant)
We looked at the cultures grown overnight and found some grew better than others. The cells growing in tubes A,B and the four controls showed good visible growth. However, the other tubes showed no growth and thus were incubated for another day.
