|The sweet smell of ...E.coli?|| Main project page|
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We began writing our proposal today. We grasped a better understanding of the vanillin pathway and the light sensing device that we will be using. We decided on a single vector system to introduce the desired genes into E.Coli. We also found a suitable E.Coli strain that exspresses higher levels of tyrosine, the starting material for the vanillin pathway. Finally, we outlined a rough procedure of the necessary steps we will need take to get our system up and running.
All parts that we will be using can be found in the registry and part numbers can found in are proposal that was submitted today.
The proposal is posted on the main project page
Red light sensitive E.coli that produces vanillin.
The aim of our project is to successfully transform E.coli to produce vanillin (vanilla smell) in the presence of red light. The project is a model for controlling protein production in living organisms using the cheap available source of light. Already available parts in the registry will be used for this purpose. The vanilla and the light sensitive pathway have been identified and all parts are available through iGEM registry. The following devices consisting of these parts will used in our project:
For the vanillin production:
* BBa_I742142: Gene SAM 8 (1536 bp) * BBa_I742141: Gene SAM 5 (1542 bp) * BBa_I742109: Gene COMT (1117 bp) * BBa_I742113: Gene Fcs (850 bp) * BBa_I742115: Gene Ech (1789 bp)
For the light sensitivity production:
* BBa_R0082 : Gene Omp R (108 bp) * BBa_M30109: Dual regulation
1. Obtain device (combined parts) for the light sensitivity and vanillin production from the registry. (Question from Axel: above, you listed parts you want to use - not devices. Are the devices in the library? If so, which numbers? If not, how will you make them? Very important: you should DESCRIBE your parts - not just list them!)
2. Combine the two devices using restriction enzymes and DNA Ligase.
3. Verify the size the of combined device using acrylamide gel electrophoresis.
4. Ligate the device in the vector pTG 262 (Registry part: BBa_I1742123)
5. Transform chemically competent E.coli strain DH5alpha with the vector containing the combined devices.
6. Make LB plates containing tyrosine and for selection, Chloramphenicol antibiotic. Plate transformed cells. (5 plates with 20ul of transformed cells and 5 plates with 200ul transformed cells)
7. Incubate plates at 37C overnight.
8. Identify positive colonies (antibiotic and red fluorescents protein) Note: If colonies fluoresces red they are negative (device will get inserted at the region that will disrupt the expression of rfp.
9. Grow positive colonies in suspension cultures of LB/Chloramphenicol overnight.
10. Plate cells and observe results.