840:153g:Projects/project2/2008/09/15
 The sweet smell of ...E.coli?
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Update of the project proposalSome minor formatting was done for the project proposal and is posted on the main project page as well as here. 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. A few questions related to the characterization of the parts used will be tackled in this project. The project is a model for controlling protein production in living organisms using a cheap available source of light. The vanillin and the light sensitive pathway have been identified and all parts are available through the iGEM registry. The following devices and parts will used in our system: * BBa_M30109: When red light is detected by the red light sensitive domain, the production of phosphorylated OmpR is disrupted. Phosphorylated OmpR activates the OmpC promoter, and this in turn inhibits the OmpF promoter. The OmpF promoter is part BBa_R0082 in the iGEM registry, and is the promoter we are using to initiate production of vanillin. Therefore, in the dark, the OmpF promoter will be inhibited by the pathway starting with phosphorylated OmpR, however; in the presence of red light, OmpR will not be phosphorylated and our chosen promoter will not be inhibited. * BBa_R0082: This part is a promoter region that is inhibited in the dark by the activation of the OmpC promoter by phosphorylated OmpR. This part will be ligated between BBa_M30109 and BBa_I742140. * BBa_I742140: Beginning with tyrosine, this device produces 4-hydroxy-3-methoxybenzaldehyde(vanillin). * BBa_I1742123: This is the vector we will be using to introduce our system to E. coli. It has an rfp domain that will be disrupted by the successful insertion of our system. E. coli containing vector that has been successfully changed will not fluoresce. This vector also has Chloramphenicol antibiotic resistance, therefore we can select for colonies that have vector containing our system and those that do not contain the vector. * EnvZ deficient E. coli: We still need to obtain E. coli deficient in native EnvZ. EnvZ is associated with the phosphorylation of OmpR. If there is native EnvZ, we cannot control our system with the red light sensitive domain because OmpR would be phosphorylated when we don't want it to be, and that will start the pathway that inhibits our promoter. The steps involved in our project: 1. Obtain the parts and devices listed above from the iGEM registry, store those parts in E. coli colonies, and locate EnvZ deficient E. coli. 2. Combine our two devices and promoter part in the order BBa_M30109, BBa_R0082, BBa_I742140. 3. Verify the size of our system using acrylamide gel electrophoresis. 4. Ligate our system into the vector pTG 262 (part: BBa_I1742123 listed above). 5. Transform chemically competent EnvZ deficient E.coli strain with the vector containing the combined devices. 6. Make LB plates containing varying tyrosine concentration 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 fluoresce red they are negative. 9. Grow positive colonies in suspension cultures of LB/Chloramphenicol overnight to kill those colonies that do not contain our vector. 10. Test functionality of our system. We will attempt to answer a number of questions with our system: * First, we will test to see if our system is working as intended, i.e. vanillin is produced when red light is shined on the colonies and that production stops when the colonies are in the dark. * Second, we will test whether or not tyrosine is needed in the growth media which would be done by growing positive colonies on plates with varying concentrations of tyrosine. * Third, we will attempt to determine the amount of tyrosine needed in the growth media to optimize production of vanillin. * Fourth, we will attempt to determine the amount of time it takes for our system to begin producing vanillin after red light is introduced and the time it takes for the vanillin production to stop when the light is turned off.
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