Part:BBa_K2995002
Part Improvement: BBa_K2573000
The original BioBrick part can be found here: https://parts.igem.org/Part:BBa_K2573000
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1835
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2638
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. Therefore, we recommend integrating this part into E. coli's genome. This can be accomplished by cloning the biobrick into a miniTn7 vector. See the iGEM registry's genome integration toolkit (https://parts.igem.org/Genome_Integration) for details.
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, we improved this part by making upon the MetE inducible. 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.
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 to show the ability for of our new system to be controlled with light.
Two strains of JT2 were tested in order to characterize the function of the new BioBrick (BBa_K2995002). Both strains were grown for 6 hours under 4 different conditions: M9 with methionine under green light, M9 without methionine under green light, M9 with methionine under red light and M9 without methionine under green light.
1. JT2 with BBa_K2573000 in genome
Tested in order to confirm the function of the improved BioBrick BBa_K2995002. 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 indicates the proper function of this brio brick. The absence of growth in either condition or growth in both conditions would indicate improper function of this biobrick.
2.Empty JT2 (no metE gene)
Used as a negative control for growth in media without methionine and a positive control for growth in media with methionine (control for not 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 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. Moreover the previous characterization of the original part BBa_K2573000 the need for inducible control of the metE gene. Therefore, BBa_K2995002 has been improved by allowing 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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