Part:BBa_K2573000
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.
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
- 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 1864
- 1000COMPATIBLE WITH RFC[1000]
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:
| Sample | OD 600nm |
|---|---|
| JT2 Empty (+ meth) | 0.011 |
| JT2 Empty | 0.012 |
| BBa_K2573000 (+meth) | 0.011 |
| BBa_K2573000 | 0.010 |
| BBa_J04450 (+meth) | 0.005 |
| BBa_J04450 | 0.006 |
Figure 3: Growth of each strain in M9 with or without methionine
OD 600 after 10h of growth in 37 degree incubator
| Sample | OD 600nm | Growth |
|---|---|---|
| JT2 Empty (+ meth) | 1.262 | Positive |
| JT2 Empty | 0.0752 | Negative |
| BBa_K2573000 (+meth) | 1.237 | Positive |
| BBa_K2573000 | 1.311 | Positive |
| BBa_J04450 (+meth) | 1.420 | Positive |
| BBa_J04450 | 1.506 | Positive |
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
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. 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... 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.
2. Homology arms were added to the sequence to facilitate the replacement of an existing MetE gene, allowing for the device's integration into prototrophic strains for methionine.
the documentation of your improvement (text, graphs, pictures, tables, etc.) Include sections for purpose, results, methods, etc. (if applicable) Uploads: (links to uploads relevant to your contribution, ex: csv containing your data, sequence files, etc.) Note: All files pertaining to a contribution must be uploaded to the Registry.
//plasmid
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