Difference between revisions of "Part:BBa K1587007"
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<partinfo>BBa_K1587007 short</partinfo> | <partinfo>BBa_K1587007 short</partinfo> | ||
− | Strong RBS | + | ===BioBrick Description=== |
+ | |||
+ | Strong RBS: [https://parts.igem.org/Part:BBa_B0030 BBa_B0030], enzymes: pyruvate formate-lyase 1 pflB and pyruvate formate-lyase 1-activating enzyme pflA and terminator: [https://parts.igem.org/Part:BBa_B1006 BBa_B1006] (Figure 1). | ||
<center>[[File:TLSE For.jpg]]</center> | <center>[[File:TLSE For.jpg]]</center> | ||
− | <center>'''Figure 1: Genetic | + | <br> |
+ | <center>'''Figure 1: Genetic construction for formate production.'''</center> | ||
+ | <br> | ||
+ | |||
+ | |||
+ | ===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:<br> | 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.<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. | + | - '''''pflA''''' coding for pyruvate formate lyase activase, which is directly linked with the pyruvate formate lyase, enabling its activation (Figure 2). |
<center>[[File:TLSE ForMetabo.jpg]]</center> | <center>[[File:TLSE ForMetabo.jpg]]</center> | ||
+ | <br> | ||
<center>'''Figure 2: Metabolic pathway to produce formate via glucose using BBa_K1587007 in ''E. coli''.'''</center> | <center>'''Figure 2: Metabolic pathway to produce formate via glucose using BBa_K1587007 in ''E. coli''.'''</center> | ||
+ | <br> | ||
+ | |||
− | |||
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− | |||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K1587007 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1587007 SequenceAndFeatures</partinfo> | ||
+ | <br> | ||
+ | Sequencing has been made thanks to 4 specific primers for genes contained in BBa_K1587007 in order to have the complete sequence: <br> | ||
+ | - Forward: pflB, pflA & VF2<br> | ||
+ | - Reverse: pflB<br> | ||
+ | So, sequence is <b>validated</b>. | ||
+ | |||
+ | |||
+ | |||
+ | ===Experiments=== | ||
+ | |||
+ | In order to test BBa_K1587007, we add a constitutive promoter p(Bla): [https://parts.igem.org/Part:BBa_I14018 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].<br> | ||
+ | Our main [http://2015.igem.org/Team:Toulouse/Results#formaproduct result] is shown by the graphic below: | ||
+ | |||
+ | <center>[[File:TLSE ForProd.PNG]]</center> | ||
+ | <br> | ||
+ | <center>'''Figure 3: NMR analysis of formate production using BBa_K1587007 after 3 days culture under micro-aerobic condition'''</center> | ||
+ | <br> | ||
+ | |||
+ | Formate production increased significantly by <b>10 %</b> compared to wt with BBa_K1587007 in ''E. coli'' (Figure 3). | ||
+ | |||
+ | |||
Latest revision as of 18:49, 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).
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).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 405
Illegal BamHI site found at 2807 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 514
- 1000INCOMPATIBLE 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:
Formate production increased significantly by 10 % compared to wt with BBa_K1587007 in E. coli (Figure 3).