Difference between revisions of "Part:BBa K2573000"

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===Usage and Biology===
 
===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.
  
−
Methionine is an essential amino acid for E.coli growth. Respectively, the E. coli MetE gene is essential to proper methionine synthesis. It encodes for an enzyme that catalyzes the final step of de novo methionine biosynthesis without using an intermediate methyl carrier. The absence of vitamin B12 is also required, as it functions as a repressor of MetE.
+
[[File:Biosynthesis-of-methionine-A-Methionine-synthase-MetH-MetE-reaction-B.png|300px|thumb|left|Biosynthesis of Methionine Mechanism]]
  
−
"https://www.researchgate.net/figure/Biosynthesis-of-methionine-A-Methionine-synthase-MetH-MetE-reaction-B_fig8_276507951"
 
  
 +
This part is the MetE gene cassette under the inducible LacI promoter, designed to be a MetE coding device.
  
−
This part is the MetE gene cassette under the inducible LacI promoter. A 2.4kb fragment containing the MetE gene from plasmid pSK397 was cloned into bba_0J4450 via PCR and NEBuild Hi-Fi Assembly.  
+
A 2.4kb fragment containing the MetE gene from plasmid pSKA397 was cloned into Bba_J04450 via PCR and NEBuilder HiFi DNA Assembly.  
  
  
Line 33: Line 34:
  
 
===Characterization of MetE Coding Device===
 
===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.
 +
 +
 +
[[File:UWaterloo BioBrick characterization labelled.png|500px]]
 +
 +
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.
 +
 +
 +
[[File:UWaterloo BioBrick characterization labelled 2.png|500px]]
 +
 +
Figure 2: Each strain (BioBrick in JT2, empty JT2, DH5a) was inoculated in M9 with and without methionine.
 +
 +
 +
<ins>OD 600 after inoculation</ins>
 +
 +
- 20ul of overnight culture (washed of LB) inoculated into 5ml M9 (with or without methionine) and appropriate antibiotics:
 +
 +
<table>
 +
<tr>
 +
<th style="width: auto;">Sample</th>
 +
<th style="width: auto;">OD 600nm</th>
 +
</tr>
 +
<tr>
 +
<td>JT2 Empty (+ meth) </td>
 +
<td>0.011 </td>
 +
</tr>
 +
<tr>
 +
<td>JT2 Empty</td>
 +
<td>0.012 </td>
 +
</tr>
 +
<tr>
 +
<td>BBa_K2573000 (+meth) </td>
 +
<td>0.011 </td>
 +
</tr>
 +
<tr>
 +
<td>BBa_K2573000  </td>
 +
<td>0.010</td>
 +
</tr>
 +
<tr>
 +
<td>BBa_J04450 (+meth) </td>
 +
<td>0.005 </td>
 +
</tr>
 +
        <tr>
 +
<td>BBa_J04450  </td>
 +
<td>0.006 </td>
 +
</tr>
 +
</table>
 +
 +
 +
 +
[[File:Biobricksfinalcharacterization.png|500px]]
 +
 +
Figure 3: Growth of each strain in M9 with and without methionine
 +
 +
 +
 +
<ins>OD 600 after 10h of growth in 37 degree incubator</ins>
 +
 +
<table>
 +
<tr>
 +
<th style="width: auto;">Sample</th>
 +
<th style="width: auto;">OD 600nm</th>
 +
                <th style="width: auto;">Growth</th>
 +
</tr>
 +
<tr>
 +
<td>JT2 Empty (+ meth) </td>
 +
<td>1.262 </td>
 +
<td>Positive </td>
 +
</tr>
 +
<tr>
 +
<td>JT2 Empty</td>
 +
<td>0.0752 </td>
 +
<td>Negative</td>
 +
</tr>
 +
<tr>
 +
<td>BBa_K2573000 (+meth) </td>
 +
<td>1.237 </td>
 +
<td>Positive </td>
 +
</tr>
 +
<tr>
 +
<td>BBa_K2573000  </td>
 +
<td>1.311</td>
 +
<td>Positive </td>
 +
</tr>
 +
<tr>
 +
<td>BBa_J04450 (+meth) </td>
 +
<td>1.420 </td>
 +
<td>Positive </td>
 +
</tr>
 +
        <tr>
 +
<td>BBa_J04450  </td>
 +
<td>1.506 </td>
 +
<td>Positive </td>
 +
</tr>
 +
</table>
 +
 +
 +
<b>RESULTS</b>
 +
 +
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.
 +
 +
===Need For Improvement===
 +
 +
Group: iGEM19_Waterloo (2019-10-21)
 +
 +
We conducted the following experiment to show that a change in the promoter sequence of this part is necessary since we suspected that the current LacI promoter was not inducible in  the strain JT2.
 +
 +
--IPTG-Inducibility Test:--
 +
 +
The JT2 strain with BBa_K2573000  was grown in fully supplemented M9 supplemented. Two samples were grown with 1 mM IPTG included in the medium, and two samples were grown without IPTG. Four OD readings were taken for each sample between OD 0.05 and OD 0.25 to ensure that all readings were comfortably within the region of exponential growth. By taking the logarithm of this OD data, performing a linear fit, and taking the inverse of the slope of the line, we obtained doubling times for the samples.
 +
 +
The two samples grown without IPTG had doubling times of 91.0 minutes and 92.0 minutes. The two samples grown with IPTG had doubling times of 92.5 minutes and 99.1 minutes. Had there been a significant change in protein production upon IPTG addition, there should have been a substantial metabolic load that would have resulted in a large change in doubling time. Since the change in doubling time was minimal, one can infer that the addition of IPTG has little if any effect. This, in conjunction with the fact that JT2 with BBa_K2573000 can survive in media without IPTG and methionine (seen in original characterization of this part), means that the current construct is not meaningfully inducible. It is therefore logical to change the promoter on the MetE gene.
 +
 +
===Part Improvement===
 +
 +
Group: iGEM19_Waterloo    (2019-10-21)
 +
 +
Author: Michael Lam, Katie Walker, Clara Fikry, Leah Fulton
 +
 +
<b>Summary</b>
 +
 +
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.
 +
 +
[[File:CcaS-R.png|400px|thumb|centre|Induction Mechanism of CcaS/R Promoter [2]]]
 +
 +
 +
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)
 +
 +
<b>RESULTS</b>
 +
 +
[[File:Empty JT2.png|500px]]
 +
 +
 +
Figure 4: Empty JT2 inoculated in M9 with and without methionine.
 +
 +
 +
<ins>Growth of Empty JT2 in M9</ins>
 +
 +
<table>
 +
<tr>
 +
<th style="width: auto;">Sample</th>
 +
                <th style="width: auto;">Light</th>
 +
                <th style="width: auto;">Growth</th>
 +
</tr>
 +
<tr>
 +
<td>JT2 Empty (+ meth)  </td>
 +
<td>Green  </td>
 +
<td>Positive </td>
 +
</tr>
 +
<tr>
 +
<td>JT2 Empty</td>
 +
<td>Green  </td>
 +
<td>Negative</td>
 +
</tr>
 +
<tr>
 +
<td>JT2 Empty (+ meth)  </td>
 +
<td>Red  </td>
 +
<td>Positive</td>
 +
</tr>
 +
<tr>
 +
<td>JT2 Empty</td>
 +
<td>Red  </td>
 +
<td>Negative</td>
 +
</tr>
 +
</table>
 +
 +
 +
 +
 +
[[File:R.png|500px]]
 +
 +
 +
Figure 5: JT2 with CcaS/R promoter inoculated in M9 with and without methionine.
 +
 +
 +
<ins>Growth of JT2 with CcaS/R Promoter</ins>
 +
 +
<table>
 +
<tr>
 +
<th style="width: auto;">Sample</th>
 +
<th style="width: auto;">Light</th>
 +
                <th style="width: auto;">Growth</th>
 +
</tr>
 +
<tr>
 +
<td>JT2 + CcaS/R (+ meth)  </td>
 +
<td>Green  </td>
 +
<td>Positive </td>
 +
</tr>
 +
<tr>
 +
<td>JT2 + CcaS/R </td>
 +
<td>Green  </td>
 +
<td>Positive </td>
 +
</tr>
 +
<tr>
 +
<td>JT2 + CcaS/R (+ meth)  </td>
 +
<td>Red  </td>
 +
<td>Positive </td>
 +
</tr>
 +
<tr>
 +
<td>JT2 + CcaS/R </td>
 +
<td>Red  </td>
 +
<td>Negative </td>
 +
</tr>
 +
</table>
 +
 +
 +
<b>Conclusion</b>
 +
 +
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.

Latest revision as of 19:14, 20 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.


UWaterloo BioBrick characterization labelled.png

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.


UWaterloo BioBrick characterization labelled 2.png

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


Biobricksfinalcharacterization.png

Figure 3: Growth of each strain in M9 with and 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.

Need For Improvement

Group: iGEM19_Waterloo (2019-10-21)

We conducted the following experiment to show that a change in the promoter sequence of this part is necessary since we suspected that the current LacI promoter was not inducible in the strain JT2.

--IPTG-Inducibility Test:--

The JT2 strain with BBa_K2573000 was grown in fully supplemented M9 supplemented. Two samples were grown with 1 mM IPTG included in the medium, and two samples were grown without IPTG. Four OD readings were taken for each sample between OD 0.05 and OD 0.25 to ensure that all readings were comfortably within the region of exponential growth. By taking the logarithm of this OD data, performing a linear fit, and taking the inverse of the slope of the line, we obtained doubling times for the samples.

The two samples grown without IPTG had doubling times of 91.0 minutes and 92.0 minutes. The two samples grown with IPTG had doubling times of 92.5 minutes and 99.1 minutes. Had there been a significant change in protein production upon IPTG addition, there should have been a substantial metabolic load that would have resulted in a large change in doubling time. Since the change in doubling time was minimal, one can infer that the addition of IPTG has little if any effect. This, in conjunction with the fact that JT2 with BBa_K2573000 can survive in media without IPTG and methionine (seen in original characterization of this part), means that the current construct is not meaningfully inducible. It is therefore logical to change the promoter on the MetE gene.

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. 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.

Induction Mechanism of CcaS/R Promoter [2]


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

Empty JT2.png


Figure 4: 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



R.png


Figure 5: 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.