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From 2010.igem.org

Phototaxis

Progress report:

Today we've designed primers and worked out how we plan to characterize our light sensor. The experiment has to determine how much our protein stimulates the kinase activity of CheA, and in turn the phosphorylation of CheY. We plan to measure directly on CheY.

Eksperiment:

- first we create a suspension of cells in water, keeping them in the dark until the experiment.

- Then we take small microcapillary tubes, and use them to draw a specific amounts of fluid and bacteria from the suspension, as to ensure equal amounts of protein in our gels, more on which later.

- We will then ad radio labeled ATP to our cells. (This step might be done earlier)

- We now shine a colored light into our tube, varying wavelength and intensity in separate experiments.

- After an equal length of time for each tube, we lyse the cells, and run the proteins through a gell.

- We might need to tag our CheY with antibodies to show them, but in theory the only proteins that should increase in phosphorylation states would be CheY and CheA, and therefore the remaining bands should stay unchanged.

We hope to show how the kinase activity of CheA on CheY is stimulated by our blue light, which wavelengths are optimal, and whether varying intensity of light has an effect on activity.

BioBrick Design: We designed and ordered the following primers:
// Melting points calculated with Oligocalc [http://www.basic.northwestern.edu/biotools/oligocalc.html]

NpSRII-HtrII-EcTsr primers:
Upstream primer:
5' - GTTTCTTCGAATTCGCGGCCGCTTCTAGATGGTGGGACTTACG - 3' (Melting point basic: 71°C)

Downstream primer:
5' - GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTAAAATGTTTCCCAGTTCTCCTCG - 3' (Melting point basic: 71°C)

ninaB primers (Retinal enzym):
Upstream primer:
5' - GTTTCTTCGAATTCGCGGCCGCTTCTAGATGGCAGCCGGTGTCTTCAA - 3' (Melting point basic: 73°C)

Downstream primer:
5' - GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTACTAAATGGCATTGGGTGCAA - 3' (Melting point basic: 71°C)

References:
OligoCalc: http://www.basic.northwestern.edu/biotools/oligocalc.html

Flagella

Today we have designed mutation primers, to remove a Pst1 site inside our operon. We have planned a silent mutation, so if we get it right it shouldn't affect our final protein.

Biobrick design:

FlhDC mutationsprimers:
Passer til komplimentærstrengen:
5' - GGCAGCTTTGCCCGCAGCTTATGTC - 3' (Melting point basic: 63°C)

Passer til coding strand:
5' - GACATAAGCTGCGGGCAAAGCTGCC - 3' (Melting point basic: 63°C)

--CKurtzhals 20:04, 5 July 2010 (UTC)

Modelling:

Progress report: Today we have been discussing Stokes and Navier-Stokes equations in relation to the system we would like to model. After witch we tried to identify a differential equation describing the system. The following physics books have been used

The theory of polymer dynamics, M. DOI, S. F. Edwards, oxford science publications, 4. edition 1992.

Physics of continuous matter, B. Lautrup, IOP publishing, 2005.

Working hypothesises: a row of rigid screws with one end attached to a surface with the ability to bend in an angel θi and generate a force Fi. The forces generated by a screw may influence the angel of other screws and the effect the entire system witch in term affects the first screw. When the physics of the system is determined we hope to model variations of θi with the force Fi.

--Toand 19:06, 5 July 2010 (UTC)