840:153g:Projects/project2

Team Members

• Jared Koelling [[1]]
• Matt Schneider [[2]]
• Corey Hartbecke [[3]]
• Surabhi Gupta [[4]]
• Aaron Miller [[5]]
• Diveena Vijayendran [[6]]

Image:http://www.openwetware.org/wiki/Image:Picture_005.jpg Image:http://www.openwetware.org/wiki/Image:Recomb_DNA_fall_08_.jpg

Project Description

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:

• • Part BBa_I1742123:
• 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:

• BBa_M30109,BBa_R0082,BBa_I742140.

[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.

Our Team Journal

On the calender above, select the desired date and read our progress on the project.

Need to Know

Can we get the desired E.coli strain.

Project Summary

Summary of Making our Part: We initially proposed to attach a red light sensitive promoter to a vanilla enzymatic pathway. However, just before starting our project we discovered that our vanilla parts were not actually in the iGEM registry but rather they were only proposed parts. We switched our project parts to the enzymatic pathway of wintergreen production that converts salicylic acid to methyl salicilate, the compound that smells like wintergreen in addition we proposed to characterize this new composite part to determine how much salicylic acid is required and how long it take for production of methyl salicylate. We also had to switch our promoter to a UV promoter; the red light promoter required a special strain of E. coli that was deficient in an enzyme ENVz. We attempted to obtain this strain but couldn’t get it. Dr. Schwekendiek requested glycerol stock from iGEM of our part BBa_J45119 (Wintergreen part without the tetracycline promoter), part BBa_J45120 (Wintergreen part with the tetracycline promoter) and the UV promoter part BBa_i765001. This was because of several unsuccessful attempts to extract these parts from the iGEM paper library and the well plates provided. As part of our initial goal, we intended to amplify out the functional wintergreen part of BBa_J45120, without the promoter and the non functional second terminator attached to the part. The extracted part BBa_J45119, does not have a promoter region but is BioBrick compatible and thus ready to be linked with 5001, BBa_J45119 was to be used as a backup if we didn’t successfully extract the functional part from J45120. When we obtained the glycerol stocks from iGEM, we plated some cells on LB plates and grew them overnight. Colonies were picked out, overnight cultures grown, and plasmid was extracted from those colonies. The extracted plasmid was digested with EcoR1 and Spe1 to see if the parts were present in the plasmids (all BioBrick compatible have EcoR1 prefacing them and Spe1 after the part). Part 120 was expected to be 1292bp and part 5001 was expected to be 76bp. Gel electrophoresis showed a band between 1200bp and 1500bp for part 120 and no results for part 5001. Another plasmid digest was carried out for part 5001 with increased volume of plasmid. Gel electrophoresis showed a smear with no significant results. We then decided to amplify out part 5001 with M13 primers. Primers were designed to amplify out part 120 as desired with overhangs of restriction sites Xbal on the forward primer and the restriction site Spe1 on the reverse primer. Part 120 was amplified out with these primers. As expected, gel electrophoresis showed a band at 1230bp for part 120. A smear was observed for part 5001. We discovered on the iGEM website that iGEM suggests a set of forward and reverse primer to amplify out any bio-brick part from the library known as VF-2 and VR, which Dr. Schwekendiek ordered for the class use. The set of primers were used to amplify out part 5001 from the plasmid but the results were inconclusive. Restriction digestion of part 120 with restriction enzymes showed that we designed our primers to amplify out part 120 but the amplified part was not BioBrick compatible. To be bio-brick compatible it should have had restriction site Pst1 as well as Spe1 on the reverse primer to meet the prefix and suffix standards of iGEM. Therefore, a new reverse primer was designed with the correct BioBrick compatible suffix. Another plasmid extraction for part 5001 was done from overnight cultures using a minprep kit. A restriction digestion using EcoR1 and Pst1 was carried out to verify the presence of the bio-brick part 5001 in the plasmid. The expected size was 117bp and the gel electrophoresis showed a band at that region. Part 120 was PCR amplified with the old forward primer and the newly designed reverse primer. The first round of amplification did not yield a band at the expected size, lowering the annealing temperatures in the second PCR reaction yielded a band at ~1200bp on agarose gel electrophoresis.

Plasmid containing part 5001 was double digested with Spe1 and Pst1 to create sticky ends.

PCR product of part 120 was digested with Xbal and Pst1 to create sticky ends so it could be ligated behind the UV promoter. Both the digested parts were purified used Qiagen PCR purification kit. Ligation of 120 and 5001 was carried out overnight at 4C. Competent E. coli cells were transformed with the ligated plasmid and the cells were grown on LB ampicillin plates. Growth of 4 colonies was observed. Overnight cultures and plasmid miniprep were performed for all the colonies. The plasmids were digested with EcoR1 and Pst1. The plasmid containing our insert has a band at 1323bp.

2 out of the 4 colonies showed a band at approximately 1300bp (between 1000bp and 1500bp, closer to 1500bp). The composite BioBrick in the plasmids was sent out for sequencing. The sequencing results that came back did not match our expected part. The longest sequence result was 400bp instead of 1323bp, and the sequence did not significantly match our designed device. Summary of Characterizing our Parts Production of Wintergreen We tested Part 120, which was already a functional bio-brick part, for production of wintergreen smell. The cells were plated on LB + tetracycline plates. There was cell growth at concentration of tetracycline lower than 60ul of 12ug/ul but no wintergreen smell could be detected through smell testing. We found out that salicylic acid is needed as the precursor for the production of methyl salicylate, which we did not add in the first experiment. Suspension cultures of cells containing tetracycline and varying concentrations of salicylic acid were grown. A smell test survey was conducted with random people to see if they observed any significant difference in the smell of the suspension cultures. The cultures containing 0.06% salicylic acid was generally considered to have the best smell. Dr. Elbert from the chemistry department told us to carry out pH tests on the suspension cultures to determine changes in pH as salicylic acid is converted to methyl salicylate. The pH test that was performed suggested that as salicylic acid is converted to methyl salicylate, the pH increases. The next step in our project was to characterize our composite part of 5001/120 BioBrick part. A characterization experiment was designed for our part. The cells were grown in suspension cultures in petri dishes with varying exposure times to either UV light or fluorescents light. The test turned out to be negative, no wintergreen smell was detected with up to 2 days of light exposure and the pH test showed a changed to a more basic solution; however, the control plate that was not exposed to light also showed the same more basic results.

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