Difference between revisions of "Part:BBa K2573000"

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MetE Coding Device

Usage and Biology

Methionine is an essential amino acid for E.coli growth, and the MetE gene is essential for methionine synthesis. It encodes for an enzyme that catalyzes the final step of de novo methionine biosynthesis without using an intermediate methyl carrier. For optimal MetE function, vitamin B12 should not be present, as it functions as a MetE repressor.

Biosynthesis of Methionine Mechanism

This part is the MetE gene cassette under the inducible LacI promoter, designed to be a MetE coding device.

A 2.4kb fragment containing the MetE gene from plasmid pSKA397 was cloned into Bba_J04450 via PCR and NEBuilder HiFi DNA Assembly.

REFERENCES: Pejchal, Robert, and Martha L Ludwig. “Cobalamin-Independent Methionine Synthase (MetE): A Face-to-Face Double Barrel That Evolved by Gene Duplication.” PLOS ONE, Public Library of Science, journals.plos.org/plosbiology/article?id=10.1371%2Fjournal.pbio.0030031.

“Escherichia Coli K-12 Substr. MG1655 MetE.” MetaCyc Parathion Hydrolase, biocyc.org/gene?orgid=ECOLI&id=EG10584.

Sequence and Features

Characterization of MetE Coding Device

We transformed this BioBrick (BBa_K2573000) into E. coli strain JT2. JT2 has MetE in its genome, under the CcaS/R promoter. However, this strain does not contain genes encoding CcaS/R, meaning it is unable to express MetE.

BBa_K2573000 in JT2 was grown in M9 medium with and without methionine. Since methionine is essential for bacterial growth, and JT2 is a methionine knockout, growth of JT2 in a methionine deficient medium indicates that the MetE BioBrick functions as intended. Empty JT2 cells were also grown in M9 with and without methionine, to confirm that the MetE gene in the JT2 genome is non-functional. BBa_J04450 in E. coli DH5a cells, which contain MetE in the genome, were also grown in the same media as a control to show that bacteria are able to grow in the M9 prepared.

Figure 1: BioBrick in JT2, empty JT2, and DH5a cells were grown in LB. Overnight cultures of BioBrick in JT2, empty JT2, and DH5a were rinsed 3x with M9 to remove residual LB.

Figure 2: Each strain (BioBrick in JT2, empty JT2, DH5a) was inoculated in M9 with and without methionine.

OD 600 after inoculation

- 20ul of overnight culture (washed of LB) inoculated into 5ml M9 (with or without methionine) and appropriate antibiotics:

Figure 3: Growth of each strain in M9 with and without methionine

OD 600 after 10h of growth in 37 degree incubator

RESULTS

The empty JT2 was able to grow in M9 with methionine, but not able to grow in M9 without methionine, showing that JT2 is unable to grow in methionine deficient media. However, when transformed with this BioBrick, (BBa_K2573000) JT2 was then able to grow in M9 without methionine.

Part Improvement

Group: iGEM19_Waterloo (2019-10-21)

Author: Michael Lam, Katie Walker, Clara Fikry, Leah Fulton

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.

Induction Mechanism of CcaS/R Promoter [2]

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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), treatments of the E. coli strain JT2 were grown in M9 medium with and without methionine, and under exposure to either red or green light. The growth of JT2 in methionine deficient medium indicated the expression of methionine and thus the function of a BioBrick, since methionine is essential for bacterial growth and JT2 is a methionine knockout. As well, the growth of JT2 in methionine deficient medium when exposed to green light but not when exposed to red light characterized the function of the inducible CcaS/R promoter in the improved Biobrick (BBa_K2995002). Growing JT2 in M9 medium with methionine acted as a positive control for JT2 growth in each treatment.

Three treatments of JT2 were tested in order to characterize the function of the new BioBrick (BBa_K2995002):

1. Transformed the new BioBrick (BBa_K2995002) into JT2.

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. Transformed the original BioBrick (BBa_K2573000) containing the LacI promoter into JT2.

This treatment acted as a negative control for MetE gene inducibility, as JT2 transformed with BioBrick BBa_K2573000 would express methionine regardless of being exposed to green or red light.

3. Empty JT2 without transformation.

Empty JT2 cells were also grown in M9 with and without methionine as a negative control for MetE gene expression.

RESULTS

Figure 4: Empty JT2 inoculated in M9 with and without methionine.

Growth of Empty JT2 in M9

Growth of JT2 with LacI Promoter

Figure 5: JT2 with CcaS/R promoter inoculated in M9 with and without methionine.

Growth of JT2 with CcaS/R Promoter

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.