Part:BBa_K2945006

AlkL Composite

This part contains the gene for the the AlkL channel protein, a ribosome binding site, and the SigA promoter. The AlkL channel protein comes from the organism Pseudomonas putida. It functions as a passive import protein that moves alkanes across the cell membrane. The purpose of this part is to eventually export long chain alkanes from our chassis organism. We chose this part for three main reasons. Firstly, the protein already has an affinity for alkanes. Our goal is to use this channel as an exporter, but the ability to facilitate alkane movement is a great first step. Secondly, the protein is prokaryotic. Eukaryotic proteins often need chaperone proteins to properly fold. By using a prokaryotic protein, we can more easily work with the part without needed these chaperones. Lastly, the gene is relatively short, allowing for easier transformation into E. coli.

Growth with the Exporter (AlkL) operon seems to follow that of the control quite closely. The Alk Operon, however, ends at a lower plateau, which is likely due to alkane toxicity. Growth with both operons plateaus with an even lower optical density. A possible reason for this lower plateau is the presence of two antibiotics (ampicillin and chloramphenicol).

This secondary growth curve test compares cells with both Alk and AlkL operons to control cells with empty vectors with both antibiotic resistances. The cells with both operons reach a lower plateau than that of the control cells. This suggests that despite being an importer, AlkL's passivity does allow for alkanes to exit the cells when the intracellular concentration reaches a high enough point. For the Alk+AlkL cells though, this point is reached when the alkane levels within the cell are already too toxic, and growth has slowed.

The AlkL gene was FLAG-tagged and expression was tested in E. coli. The expected protein size is 26 kDa. The Western Blot showed the proper band size, showing that the AlkL channel protein was successfully expressed in E. coli. This result confirms that AlkL channel is present in the E. coli in the growth curve tests above.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 186
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 680
  
• 1000
  COMPATIBLE WITH RFC[1000]