Part:BBa_K2995002
Part Improvement: BBa_K2573000
The original BioBrick part can be found here: https://parts.igem.org/Part:BBa_K2573000
Summary
The MetE coding device was designed to be implemented in an auxotrophic strain of E.coli for methionine, or a strain that is incapable of biosynthesizing methionine on its own. The system does not function within a plasmid, and must be integrated into the genome [1]. The integration of this device allows methionine, an essential amino acid for E. coli growth, to be synthesized endogenously. The coding device was comprised of the MetE gene cassette under the LacI promoter. However, the LacI promoter originally used was not inducible in JT2. Therefore, two improvements upon the MetE Coding Device were made:
1. The LacI promoter was swapped out for the optogenetically controllable CcaS/R promoter.
The new MetE coding device has the inducible CcaS/R promoter in place of the LacI promoter, allowing for the expression of methionine to be controlled optogenetically. With the CcaS/R promoter, exposure to green wavelengths of light activate gene expression, while exposure to red wavelengths of light turn off gene expression.
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REFERENCES:
[1] Milias-Argeitis A, Rullan M, Aoki SK, Buchmann P, Khammash M. Automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth. Nat Commun. 2016;7:12546. Published 2016 Aug 26. doi:10.1038/ncomms12546
[2] CcaS/CcaR. OptoBase. https://www.optobase.org/switches/Cyanobacteriochromes/CcaS-CcaR/.
Characterization of Improved MetE Coding Device
Similar to the characterization done for the original BioBrick (BBa_K2573000), samples of the E. coli strain JT2 (methionine knockout) were grown in M9 medium with and without methionine, and under exposure to either red or green light.
The growth of JT2 with BBa_K2573000 (in genome) in methionine deficient medium under green light and not red indicates expression of methionine and thus the function of a BioBrick, since methionine is essential for the growth of JT2. Growing JT2 without BBa_K257300 in M9 medium with methionine acted as a positive control for JT2 growth and a negative control in samples without methionine.
Three treatments of JT2 were tested in order to characterize the function of the new BioBrick (BBa_K2995002):
1. JT2 with BBa_K2573000 in genome
This treatment was tested in order to confirm the function of the improved BioBrick BBa_K2995002. The growth of JT2 in M9 medium without methionine under exposure to green light, in combination with the absence of growth under exposure to red light would yield a positive result. The absence of growth in either condition or growth in both conditions would yield a negative result.
2. Empty JT2 (no metE gene).
Empty JT2 cells were also grown in M9 with and without methionine as a negative control for MetE gene expression.
RESULTS
Figure 1: Empty JT2 inoculated in M9 with and without methionine.
Growth of Empty JT2 in M9
| Sample | Light | Growth |
|---|---|---|
| JT2 Empty (+ meth) | Green | Positive |
| JT2 Empty | Green | Negative |
| JT2 Empty (+ meth) | Red | Positive |
| JT2 Empty | Red | Negative |
Growth of JT2 with LacI Promoter
| Sample | Light | Growth |
|---|---|---|
| JT2 + LacI (+ meth) | Green | Positive |
| JT2 + LacI | Green | Positive |
| JT2 + LacI (+ meth) | Red | Positive |
| JT2 + LacI | Red | Positive |
Figure 2: JT2 with CcaS/R promoter inoculated in M9 with and without methionine.
Growth of JT2 with CcaS/R Promoter
| Sample | Light | Growth |
|---|---|---|
| JT2 + CcaS/R (+ meth) | Green | Positive |
| JT2 + CcaS/R | Green | Positive |
| JT2 + CcaS/R (+ meth) | Red | Positive |
| JT2 + CcaS/R | Red | Negative |
Conclusion
The empty JT2 was able to grow in M9 with methionine, but not in M9 without methionine, indicating that JT2 is unable to express methionine endogenously and this lack of expression is not affected by light. JT2 transformed with the original BioBrick BBa K2573000 was able to grow in methionine deficient medium under exposure to both green and red light, indicating the noninducible nature of LacI in JT2. Moreover, JT2 was able to grow in methionine deficient medium under exposure to green light, but not under red light. This indicates that JT2 with the CcaS/R promoter is able to be optogenetically controlled, with green light activating expression of methionine and red light switching off expression. Therefore, the improved BBa_K2995002 allows for inducible methionine biosynthesis. This part can be transformed into a methionine autotroph, such as E. coli JT2, along with the CcaS/R system. Light can then be used to control growth of this engineered strain.
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