Difference between revisions of "Team:Aachen/Lab/Methanol/Labeling Experiment"

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=Summary=
 
=Summary=
  
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A batch fermentation with {{sup|13}}C labeled methanol as an additional carbon source was done to demonstrate the functionality of our constructed methanol assimilation pathway via mass spectrometry.  
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A batch fermentation with {{sup|13}}C labeled methanol as an additional carbon source was done to demonstrate the functionality of our methanol assimilation pathway. Afterwards, {{sup|13}}C labeled carbon atoms were measured  via mass spectrometry.  
  
 
Two constructs that are derived from BioBricks were tested in ''E. coli'' BL21 Gold (DE3).  
 
Two constructs that are derived from BioBricks were tested in ''E. coli'' BL21 Gold (DE3).  
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{{Team:Aachen/Figure|Aachen_MCC.png|title=Methanol Condensation Cycle scheme|subtitle=Sugars are the first metabolites that will contain {{sup|13}}C if the pathway works successfully. The initial step is the assembly of formaldehyde.|size=small}}
 
{{Team:Aachen/Figure|Aachen_MCC.png|title=Methanol Condensation Cycle scheme|subtitle=Sugars are the first metabolites that will contain {{sup|13}}C if the pathway works successfully. The initial step is the assembly of formaldehyde.|size=small}}
  
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One of them was the polycistronic expression cassette in a pSB1KRDP backbone for methanol assimilation and the other tested construct was the ''mdh'' expressed behind a IPTG inducable T7 promoter in pSB1A30.  
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One of them was the [[/Team:Aachen/Lab/Methanol/Polycistronic_Expression_Plasmid|polycistronic expression cassette]] and the other tested strain expressed the ''mdh'' behind a IPTG inducable T7 promoter in pSB1A30.  
  
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Different amounts of methanol were added to each reactor to make the polycistronic construct comparable with itself to genarate more independ data.
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Different amounts of methanol were added to each reactor to make the polycistronic construct comparable with itself to generate more independ data.
  
 
* Offgas analysis of one cultivation with the ''mdh'' construct was done constantly during the fermentation. Ideally, it detects labled carbondioxide which is produced via the detoxification pathway of ''E. coli'' as a response of the formaldehyde produced by the Mdh.
 
* Offgas analysis of one cultivation with the ''mdh'' construct was done constantly during the fermentation. Ideally, it detects labled carbondioxide which is produced via the detoxification pathway of ''E. coli'' as a response of the formaldehyde produced by the Mdh.

Revision as of 15:56, 18 September 2015


This really cool 13C labeling experiment was done at the research center 'Forschunszentrum Jülich', where Prof. Dr. Wiechert offered all necessary equipment and components to us. Jannick Kappelmann instructed two iGEM Team Aachen members (Jonas R. & Tobias S.) during the whole experiment.

Summary

A batch fermentation with 13C labeled methanol as an additional carbon source was done to demonstrate the functionality of our methanol assimilation pathway. Afterwards, 13C labeled carbon atoms were measured via mass spectrometry.

Two constructs that are derived from BioBricks were tested in E. coli BL21 Gold (DE3).

Aachen MCC.png
Methanol Condensation Cycle scheme
Sugars are the first metabolites that will contain 13C if the pathway works successfully. The initial step is the assembly of formaldehyde.

One of them was the polycistronic expression cassette and the other tested strain expressed the mdh behind a IPTG inducable T7 promoter in pSB1A30.

Different amounts of methanol were added to each reactor to make the polycistronic construct comparable with itself to generate more independ data.

  • Offgas analysis of one cultivation with the mdh construct was done constantly during the fermentation. Ideally, it detects labled carbondioxide which is produced via the detoxification pathway of E. coli as a response of the formaldehyde produced by the Mdh.
  • The samples for analysis of cytosolic metabolites were taken after one doubling time (ca. 2:25 h).
  • The analysis was done by mass spectrometry of fructose-bis-phosphate, glucose-6-phosphate and ribose-5-phosphate.

The MS-detection of cytosolic metabolites covered additionally DHAP, Xylulose-5-phosphate, Fructose-6-phosphate, E4P, 2Phosphoglycerat/3Phosphoglycerate, S7P, AMP & ADP. But these metabolites were not analysed because of natural isotope distributions, coelutions or too low concentrations.

Aachen Reactors overview 13C.jpg
Experimental Setup
Four 200 ml reactors for cultivation at 13C experiment.

Achievements

  • Proving that methanol is not assimilated into the central metabolism of E. coli BL21 Gold (DE3) when the bacteria carries the polycistronic plasmid of the four missing enzymes to complete the MCC.
  • Despite no detectable production of 13C carbondioxide in the offgas we showed significant Mdh activity with samples from the respective reactor (see Mdh Characterization)

Expected Results

3 different sugars were analyzed: fructose-bis-phosphate, glucose-6-phosphate and ribose-5-phosphate.

  • Expected mass shift if 13C,d4 is assimilated: methanol - m+5, formaldehyde - m+3
    • all other sugars in the subsequent pathway should have a shift of m+3 in the first cycle of the functional MCC.
  • Expected mass shift if 13C is assimilated: methanol - m+1, formaldehyde - m+1
    • all other sugars in the subsequent pathway should have a shift of m+1 in the first cycle of the functional MCC.

Results

In no sample of the polycistronic clones was a mass shift of m+3 was observable. The detected peak areas are similar to the analytic standard samples of 12C carbohydrates. The mass shifts m+1 & m+2 are as frequent as it is usual in the natural composition.

Aachen 13C analytstanards plot.png
Plot of natural FBP, G6P & R5P as control
This figure shows the natural mass shift composition of the measured metabolites.
  • The same is valid for reactor 6 with (#AW9K#) our polycistronic construct with 0.3 M methanol.

Conclusion & Discussion

It was not possible to show a completely working "Methanol Condensation Cycle" [1] (MCC) in E. coli.

Although no labeled cytosolic metabolites could be detected we proved qualitatively the functionality of the Mdh. This leads to the assumption that the activity of the Mdh is too low to incorporate enough formaldehyde. Another possible explanation is that subsequent enzymes of the pathway do not function properly. The measured metabolites give no hint which of the implemented steps of the MCC does not work because the measured carbohydrates are only labeled if the assimilated molecule has passed the whole MCC.

The Xpk should have no influence on the assimilation of formaldehyde because without this enzyme the pathway equals the Ribulose-Monophosphate-Pathway (RuMP) which was already shown as a functional pathway if it is implemented in E. coli[2].


Laboratory Notebook

References

  1. ↑ Bogorad IW, Chen CT, Theisen MK, Wu TY, Schlenz AR, Lam AT, Liao JC. Building carbon-carbon bonds using a biocatalytic methanol condensation cycle. Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):15928-33. doi: 10.1073/pnas.1413470111. Epub 2014 Oct 29. PubMed PMID: 25355907; PubMed Central PMCID: PMC4234558.
  2. ↑ 1. Metab Eng. 2015 Mar;28:190-201. doi: 10.1016/j.ymben.2014.12.008. Epub 2015 Jan 14.Engineering Escherichia coli for methanol conversion. Müller JE(1), Meyer F(1), Litsanov B(1), Kiefer P(1), Potthoff E(1), Heux S(2), Quax WJ(3), Wendisch VF(4), Brautaset T(5), Portais JC(2), Vorholt JA(6).

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