Part:BBa_K1587005

Butyrate synthesis pathway (BBa_1587004) without ccr gene regulated by constitutive promoter p(Bla)

Constitutive promoter p(Bla). 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 BBa_B0030. 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. This construction without the ccr gene is a negative control of the butyrate production in E. coli. Moreover, it was aimed to be inserted in our regulation system. This construction may be used in order to produce all the intermediates of butyrate up to crotonyl-CoA.

Sequencing has been made thanks to 7 forward specific primers for genes contained in BBa_K1587004 in order to have the complete sequence: VF2, hbd, crt, testB, atoB and VR Sequence is therefore validated.

Figure 1: Genetic construction to produce butyrate via glucose

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 352
  Illegal BamHI site found at 2269
  Illegal XhoI site found at 42
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1535
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 387
  Illegal BsaI.rc site found at 804
  Illegal BsaI.rc site found at 1872
  Illegal SapI.rc site found at 1884