The hybrid promoters arrived and immediately we got to work on them. There were two promoters. We ordered two hybrid promoters composed of a mammalian and bacterial promoter which both reacts to NO. The difference in our two promoters was the orientation of the bacterial and mammalian components. We labelled them 1 (mammalian + bacterial-E9-ns2 - CArG + BHN + BBaK216005) and 2 (bacterial + mammalian - BBaK216005 + BHN + CArG - E9-ns2). They came in the dry form, which we hydrated and transformed.
+
The hybrid promoters arrived and immediately we got to work on them. There were two promoters. We ordered two hybrid promoters composed of a mammalian and bacterial promoter which both reacts to NO. The difference in our two promoters was the orientation of the bacterial and mammalian components. We labelled them M-B (mammalian + bacterial-E9-ns2 - CArG + BHN + BBaK216005) and B-M (bacterial + mammalian - BBaK216005 + BHN + CArG - E9-ns2). They came in the dry form, which we hydrated and transformed.
The plasmid containing the PyeaR Bio-Brick isolated a few days ago was cut using restriction enzymes. We used EcoR1 and Pst1 as these are in the suffix and prefix of the plasmid. The restricted DNA was stored in the freezer for later confirmation of success via gel electrophoresis.
The plasmid containing the PyeaR Bio-Brick isolated a few days ago was cut using restriction enzymes. We used EcoR1 and Pst1 as these are in the suffix and prefix of the plasmid. The restricted DNA was stored in the freezer for later confirmation of success via gel electrophoresis.
Where the first week was mainly introduction to the iGEM project where we had many meetings, this week was full of ideas, lab work and research. We split into teams to do lab work and research and came together at the end and start of the day to exchange information and ideas. The week was very productive in all aspects. We finally got the transformations to work after many attempts. We realise that throughout the coming weeks, there will be many challenges which we gladly will take on solve.
Day 1
Research
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.
Labs
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.
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.
Labs
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.
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 (Chloramphenicol resistant)
Day 4
Labs
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 longer. Later, it was found that tube G showed bacterial growth but the other tubes didn't.
Joy and Rachel isolated the DNA, changing the volumes used to plasmid high copy number volumes. We were elated to find that after electrophoresis, there was isolated DNA. The wells containing DNA extracted from cells with K381001 + pSB1C3 plasmids show a similar three band pattern but of different intensity. This may be a result of varying concentrations of DNA. The
DNA samples extracted from cells with plys plasmid show an identical pattern.
and 6.
Day 5
Labs
The hybrid promoters arrived and immediately we got to work on them. There were two promoters. We ordered two hybrid promoters composed of a mammalian and bacterial promoter which both reacts to NO. The difference in our two promoters was the orientation of the bacterial and mammalian components. We labelled them M-B (mammalian + bacterial-E9-ns2 - CArG + BHN + BBaK216005) and B-M (bacterial + mammalian - BBaK216005 + BHN + CArG - E9-ns2). They came in the dry form, which we hydrated and transformed.
The plasmid containing the PyeaR Bio-Brick isolated a few days ago was cut using restriction enzymes. We used EcoR1 and Pst1 as these are in the suffix and prefix of the plasmid. The restricted DNA was stored in the freezer for later confirmation of success via gel electrophoresis.
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