The Sweet Smell of.....E.coli?
As it was first day of our lab so we prepared all the stuff which is needed in our project. we divided the work, corey and Aaron made TSS buffer, Me and Matt made LB broth and LB agar. We faced some problem in our project as Jared found out that there are some parts missing for Vanillin pathway in iGEM registry. we planned to switch the smell from vanilla to wintergreen but that could also create some problem as we dont know that how promotor will going to work. We autoclaved all the glasswares and media, and we poured plates of LB agar media for our next lab.
The final version of our proposal.
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 and also characterization of the parts that will be used. The project is a model for controlling protein production in living organisms using a cheap available source of light.
The system that we wish to put together will be made up of two devices and one part from the iGEM registry:
- 1. Device BBa_I742140: Involved in vanillin production
- This device consist 5 different parts.
- Beginning with tyrosine, this device produces 4-hydroxy-3-methoxybenzaldehyde(vanillin).
2.Device BBa_M30109: Involved in light sensitivity
- This device consist of many related parts
- In the dark: Red light not detected by red light sensitive domain --> production of phosphorylated OmpR(transcription factor) --> Phosphorylated OmpR activates OmpC promoter --> inhibition of the OmpF promoter.
- In the presence of red light: OmpR is not phosporylated --> OmpC is inhibited --> OmpF promoter is no longer inhibited.
3.Part BBa_R0082: The OmpF promoter region
- Promoter that will be used to initiate production of vanillin.
- In the dark: OmpF promoter is inhibited by the pathway starting with phosphorylated OmpR.
- In the presence of red light: OmpR is not phosphorylated therefore promoter is not inhibited.
In addition to the devices and part listed above, the following parts will also be utilized in the project:
- The vector of choice to introduce our system into E. coli.
- Contains a red fluorescents protein (rfp) domain that will be disrupted by the successful insertion of our system. E. coli containing vector that has been successfully ligated with the system will not fluoresce red.
- Vector also has Chloramphenicol antibiotic selection.
One component of our project which is not available to us through iGEM registry:
- EnvZ deficient E. coli strain
- EnvZ is associated with the phosphorylation of OmpR.
- With the presence of any native EnvZ, our system could not be controlled with the red light sensitive domain (OmpR would always be phosphorylated )
- The strain is being located and requested from investigators that have worked with it.
The proposed order of arrangement of the chosen devices and parts:
[All biobricks in the iGEM library can be combined using restriction digestion and ligation method.]
The basic steps involved in our project:
1) Make competent cells (DH5 alpha) that would house the vectors containing the extracted devices and parts from the iGEM
registry. The vector that will be used to clone the biobricks is pBlueScript.
2) Obtain the parts and devices from the iGEM registry, store those parts in E. coli colonies.
- BBa_I742140; BBa_M30109; BBa_R0082; BBa_I1742123
3) Restriction digestion and ligation of devices and part in the order described above, verification using acylamide gel electrophoresis.
4) Ligate system into vector pTG 262 (BBa_I1742123), transform EnvZ deficient E.coli
5) Selection of positive colonies (colonies without red fluorescence and resistant to Chloramphenicol) and test system functionality (presence of vanilla smell)
We will attempt to answer questions related to characterization of the 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 and observing the level of vanilla smell produced by colonies at different supplemented concentrations of tyrosine.
- Third, we will attempt to determine the amount of tyrosine needed in the growth media to optimize production of vanillin. In order to answer this question, we are trying to locate a device that will accurately gauge the level of vanillin production in parts per million. If we are unable to locate such a device, we will roughly gauge this by our sense of smell.
- 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. In order to answer this question, we are trying to locate a device that will accurately gauge the level of vanillin production in parts per million, so we can detect as early as possible when vanillin production starts. If we are unable to locate such a device, we will roughly gauge this by when we first detect vanillin smell.