Difference between revisions of "Part:BBa K1587007"
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===Usage and Biology===
 
===Usage and Biology===
  
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Formate is a simple organic acid produced with an E.coli strain. The initial substrate, glucose, is decomposed into pyruvate during glycolysis, and formate is naturally synthesized thanks to two key genes:<br>
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Formate is a simple organic acid produced with an ''E.coli'' strain. The initial substrate, glucose, is decomposed into pyruvate during glycolysis, and formate is naturally synthesized thanks to two key genes:<br>
 
- '''''pflB''''' coding for pyruvate formate lyase which catalyzes the cleavage of pyruvate into C1 and C2. This enzyme is sensitive to oxygen and is only active in microaerobic or anaerobic conditions, which is the case within our device (Figure 2).<br>
 
- '''''pflB''''' coding for pyruvate formate lyase which catalyzes the cleavage of pyruvate into C1 and C2. This enzyme is sensitive to oxygen and is only active in microaerobic or anaerobic conditions, which is the case within our device (Figure 2).<br>
 
- '''''pflA''''' coding for pyruvate formate lyase activase, which is directly linked with the pyruvate formate lyase, enabling its activation (Figure 2).
 
- '''''pflA''''' coding for pyruvate formate lyase activase, which is directly linked with the pyruvate formate lyase, enabling its activation (Figure 2).
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- Reverse: pflB<br>
 
- Reverse: pflB<br>
 
So, sequence is <b>validated</b>.
 
So, sequence is <b>validated</b>.
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===Experiments===
 
===Experiments===

Revision as of 18:47, 18 September 2015

Formate production

BioBrick Description

Strong RBS: BBa_B0030, enzymes: pyruvate formate-lyase 1 pflB and pyruvate formate-lyase 1-activating enzyme pflA and terminator: BBa_B1006 (Figure 1).

TLSE For.jpg


Figure 1: Genetic construction for formate production.



Usage and Biology

Formate is a simple organic acid produced with an E.coli strain. The initial substrate, glucose, is decomposed into pyruvate during glycolysis, and formate is naturally synthesized thanks to two key genes:
- pflB coding for pyruvate formate lyase which catalyzes the cleavage of pyruvate into C1 and C2. This enzyme is sensitive to oxygen and is only active in microaerobic or anaerobic conditions, which is the case within our device (Figure 2).
- pflA coding for pyruvate formate lyase activase, which is directly linked with the pyruvate formate lyase, enabling its activation (Figure 2).

TLSE ForMetabo.jpg


Figure 2: Metabolic pathway to produce formate via glucose using BBa_K1587007 in E. coli.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 405
    Illegal BamHI site found at 2807
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 514
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2995

Sequencing has been made thanks to 4 specific primers for genes contained in BBa_K1587007 in order to have the complete sequence:
- Forward: pflB, pflA & VF2
- Reverse: pflB
So, sequence is validated.

Experiments

In order to test BBa_K1587007, we add a constitutive promoter p(Bla):BBa_I14018. Then for formate production we made [http://2015.igem.org/Team:Toulouse/Experiments#erlencult micro-aerobic culture]. Samples were analyzed by [http://2015.igem.org/Team:Toulouse/Experiments#NMR NMR].
Our main [http://2015.igem.org/Team:Toulouse/Results#formaproduct result] is shown by the graphic below:

TLSE ForProd.PNG


Figure 3: NMR analysis of formate production using BBa_K1587007 after 3 days culture under micro-aerobic condition

Formate production increased significantly by 10 % compared to wt with BBa_K1587007 in E. coli (Figure 3).