Phototaxis:
progress report: We have run colony-PCR on the cambridge BBa_K274210 brick we have attempted to transform into our cells. We have also run colony pcr on a RBS, a RFP reporter and a heat inducible promoter. All but the K274210 colonies showed bands at correct lengths, and the RFP colonies had turned bright red.
Jakob has ordered primers and cDNA from drosophila melanogaster.
Lars Christian and Maria will be working over the weekend, to make overnight cultures, and freeze cells, so we will be ready to make a new batch of competent cells next week. We need both coli TOP10 and M1655.
Troubleshooting on Cambridge brick:
We suspect our elongation time of being to short. This might explain the smear at the bottom of each well.
-The length of the part in this plasmid is 4856Bp.
-The reaction rate for this enzyme is stated as 35-100nt/sec at 72°C and 40% of that at 55°C
-we ran the elongation for 5 minutes at 55°C giving us 4200-12.000 nt/elongation.
Another problem could be the length of our part. Apparently we are opperating on the limit of what the polymerase can do, most companies stating that it will work for 3-4kb and can be pressed to 5kb if conditions are optimal. Many also suggest increasing [MgCl2] at low yields. We might also attempt that. Yet another possibility is that our colonies didn't contain any plasmids with our part.
We will be attempting new colony pcr in the coming week, with elongation set to at least 6 minutes at 55°C. Perhaps we will attempt it with two different polymerases, increasing [MgCl2] in the Taq mix.
We have considered other ways of getting physical dna for our fusion protein. Two options come to mind if we can't get it from spudich lab.
- getting it synthesized
- Maybe getting the strain with the natromonas proteins from the 2007 melbourne team.
working hypothosis: No changes
--CKurtzhals 21:23, 9 July 2010 (UTC)
Flagella
Overview of the projekt progress and process:
1) We have had problems with out PCR's, none of them work! We have spent the last week trouble-shooting to find the problem and are down to the last few possible causes. It seems now, that the most likely cause is the annealing temperature of the primers, it is too low and the differences between the FW- and the RV-primers are too grate. We hope to solve the mystery soon. When the primer problem is solved we can finally extract the FlhDC operon from the bacteria.
2) The FlhDC operon entails an internal PST1 site, so we need to induce a silent mutation. For this we need the primers mentioned yesterday. We have fixed the primer annealing temperature problem and have ordered the primers. We hope to get them medio next week.'
These two steps are the most urgent that we work on now. Later on we have to do as follows:
3) We need to insert the FlhDC operon into the psb3k3 plasmid.
4) The psb3k3-FlhDC plasmid has to be transformed into cells.
5) Detect if cells are hyperflagellated.
Progress report: Pernille, Sheila and I have worked as a group for the first time today. We have done Miniprep to extract the plasmids we want to insert our biobricks in and Chromosomal DNA PCR to test our primers and the perfect annealing temperature(documented in labnotes)
Working Hypothesis: No Change
BioBrick Design: No Change
--Louch07 16:00, 9 July 2010 (UTC)
Modelling
Progress report: We have started implementing our model. We are discussing which forces are actually effecting the system. Besides the forces produced by the flagellas, we are discussing whether the flagellas have a favored position that it would be pushed towards, when not effected by outer forces. We are also discussing whether thermal forces should be included.
Working Hypothesis: No Change.
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