Part:BBa_K1648049

Mutated Av oprF induced by J13002

Construct Map for K1648049

OprF is a transmembrane porin which allows non-specific transmission. For constructing an efficient microbial fuel cell(MFC), we manipulated the expression of oprF. oprF allows electron carriers shuttle in and out of the bacteria, the electron generator. The electrons generated by the bacteria are taken up by the incoming carrier. Reduced electron carriers transport the electrons out of the bacteria to the electrode.

According to a paper concerning the factors affecting the conformation of OprF, we found that mutation on four Cysteine to four Serine and Lysine to Glycine at 189th position (K189G; C201S; C210S; C216S; C230S) of Azotobacter vinelandii OprF will give higher possibility of open-channel conformation with high porin activity by 5 folds. With the insertion of this mutated OprF into the bacteria, the possibly of electron carrier transmission between bacteria and extracellular as well as the efficiency of MFC would be increased by five folds. BBa_K1648049 provides constitutive transcription of BBa_K1648047 including RBS, mutated oprF and R0040(double terminator).

An experiment was carried out for the comparison between the transformed bacteria with BBa_K1648048 for oprF from Azotobacter vinelandii BBa_K1648049 for mutated oprF from Azotobacter vinelandii and BBa_K1648050 for BBa_K1172501 from Germany iGEM team. For comparison,an identical promoter, J13002, which is a constitutive promoter, is chosen to add before the gene in psb1C3 for constitutive expression of different OprF in bacteria.

Double digestion of K1648049: L: DNA ladder. Lane 1-3: Recombination Template for R0040-oprF*(BBa_K1648049) with double digestion cut at EcoR1 and PstI sites, with single digestion at Pst1 site.

Comparison between the reduction of methylene blue in tetRpro-oprF and tetRpro-oprF* transformed E.coil.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 555