Difference between revisions of "Part:BBa K1587004"
Line 17: Line 17:
 
Synthesis of butyrate has interested the scientific community because it is the precursor of butanol. Indeed, butanol would be an interesting alternative fuel especially because it could be transported by present pipeline.[1]<br>
 
Synthesis of butyrate has interested the scientific community because it is the precursor of butanol. Indeed, butanol would be an interesting alternative fuel especially because it could be transported by present pipeline.[1]<br>
 
In our project we want to produce butyrate to attract mites which infected bees but thanks to this energy interest, some metabolic pathways have been designed to produce butyrate.<br>
 
In our project we want to produce butyrate to attract mites which infected bees but thanks to this energy interest, some metabolic pathways have been designed to produce butyrate.<br>
−
An Escherichia coli strain is used for its known simplicity of genetic manipulation and its adequacy with butyrate synthesis [2]. Indeed, among the five enzymes we selected to produce butyrate from acetyl-CoA, two enzymes are naturally produced by the bacteria. The following engineered butyrate pathway has been designed:
+
An ''Escherichia coli'' strain is used for its known simplicity of genetic manipulation and its adequacy with butyrate synthesis [2]. Indeed, among the five enzymes we selected to produce butyrate from acetyl-CoA, two enzymes are naturally produced by the bacteria. The following engineered butyrate pathway has been designed:
  
 
<center>[[File:TLSE ButMetabo.jpg]]</center>
 
<center>[[File:TLSE ButMetabo.jpg]]</center>
Line 32: Line 32:
 
Sequencing has been made thanks to 7 specific primers for genes contained in BBa_K1587004: <br>
 
Sequencing has been made thanks to 7 specific primers for genes contained in BBa_K1587004: <br>
 
VF2, CCR, hbd, crt, testB, atoB and VR<br>
 
VF2, CCR, hbd, crt, testB, atoB and VR<br>
−
So, sequence is <b>validated</b>.
+
Sequence was therefore <b>validated</b>.
  
  

Revision as of 18:09, 18 September 2015

Butyrate synthesis pathway regulated by constitutive promoter p(Bla)

BioBrick Description

Constitutive promoter p(Bla). Crotonyl-coA reductase ccr coding region. 3-hydroxybutyryl-CoA dehydrogenase hbd coding region. 3-hydroxybutyryl-CoA dehydratase crt coding region. Acyl-CoA thioesterase 2 tesB coding region. acetyl-CoA acetyltransferase atoB coding region. Every genes are framed by strong RBS based on Ron Weiss thesis. This operon ends with an artificial terminator according to BBa_B1006.
Due to a lack of time we decided to have this construction synthesised instead of using the assembly protocol.

TLSE SHLAGAGA.png


Figure 1: Genetic construct to produce butyrate via glucose


Usage and Biology

Synthesis of butyrate has interested the scientific community because it is the precursor of butanol. Indeed, butanol would be an interesting alternative fuel especially because it could be transported by present pipeline.[1]
In our project we want to produce butyrate to attract mites which infected bees but thanks to this energy interest, some metabolic pathways have been designed to produce butyrate.
An Escherichia coli strain is used for its known simplicity of genetic manipulation and its adequacy with butyrate synthesis [2]. Indeed, among the five enzymes we selected to produce butyrate from acetyl-CoA, two enzymes are naturally produced by the bacteria. The following engineered butyrate pathway has been designed:

TLSE ButMetabo.jpg


Figure 2: Metabolic pathway to produce butyrate via glucose using BBa_K1587004 in E. coli



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 36
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1717
    Illegal BamHI site found at 3634
    Illegal XhoI site found at 1407
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 332
    Illegal AgeI site found at 1254
    Illegal AgeI site found at 2900
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1752
    Illegal BsaI.rc site found at 2169
    Illegal BsaI.rc site found at 3237
    Illegal SapI site found at 123
    Illegal SapI.rc site found at 3249

Sequencing has been made thanks to 7 specific primers for genes contained in BBa_K1587004:
VF2, CCR, hbd, crt, testB, atoB and VR
Sequence was therefore validated.


Experiments

In order to test BBa_K1587004 for butyrate 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].

Unfortunatly NMR analysis did not yield a significant over production of butyrate in E. coli. Nevertheless, we obtained [http://2015.igem.org/Team:Toulouse/Results#butyproduct results] as the figure 3 below:

TLSE Butprod.jpg


Figure 3: NMR analysis of butyrate production in E. coli with BBa_K1587004 and wt


We observed a statistically significant change in the production of other fermentation products such as ethanol or acetate.


References

[1] Mukesh Saini, Min Hong Chen, Chung-Jen Chiang, Yun-Peng Chao. Potential production platform of n-butanol in Escherichia coli. Metabolic Engineering 27 (2015) 76–82
[2] Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T & Liao JC (2008) Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic Engineering 10: 305–311