Difference between revisions of "Part:BBa K2573000"

Revision as of 23:04, 13 October 2019

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1864
1000
COMPATIBLE 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.

Induction Mechanism of CcaS/R Promoter

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 new MetE coding device has homology arms added to each end of the sequence, allowing the device to remove any MetE genes already existing in prototrophic strains during integration. Although prototrophic strains can biosynthesize methionine endogenously, methionine expression can now be optogenetically controlled by the CcaS/R promoter.

REFERENCES: