Part:BBa_K1895005

Controlled OrpF Expression Device for Microbial Fuel Cell Use

As part of our iGEM project we worked on trying to improve the current output from a microbial fuel cell. Microbial fuel cells which use E. coli depend on a mediator, usually methylene blue to transfer electrons between the electrodes and the organisms electron transport chain. The mediator enters the cell through porins in the cell membrane and 'steals' electrons, becoming oxidised. These electrons are then carried to the cathode, which is positively charged where they are lost, allowing electrons to flow around the circuit.

By increasing the amount of mediator that can enter the cell we hope to be able to increase the electron flow rate, or current, from our microbial fuel cell. This occurs because the electrons can be moved more freely and they're movement via the mediator is less limited by the rate at which the cell can transport the mediator across the membrane.

Usage and Biology

One of the limiting factors of a microbial fuel cell is the low permeability of the cell membrane which limits mediator transport. Team Bielefeld in 2013 expressed OprF porin protein from Pseudomonas fluorescens in E.coli which increased cell membrane permeability because the porin size is much larger than E. coli's natural porins. This allows more mediator to pass through the membrane as shown below.

One of the problems the Bielefeld team reported was the [http://2013.igem.org/Team:Bielefeld-Germany/Project/Porins reduced growth] of the E. coli due to metabolic stress. This is particularly noticeable when using the T7 promoter, as in their part BBa_K1172502. Population size is important for fuel cell output, the more cells there are the more electron donors there are and so theoretically the greater the current. To overcome this issue we have changed their promoter to a pBAD promoter which is regulated by L-arabinose monosaccharide. Thus replication stress is reduced during the first part of the growth curve. This should allow the bacterial population to grow rapidly in the fuel cell, ensuring we get a large population before L-arabinose is added (this increased level of growth should be seen by a higher OD600 value closer to 4.0 (growth of wild type) after 10 hours of growth). At this point the current should then further increase due to the larger porin expression.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 920
  Illegal BamHI site found at 65
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1312
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 833