Part:BBa_K2302009

His＋Lpp-ompA＋L-FABP

this plasmid expresses a fatty acid binding protein that can be anchoredas a fused protein on the outer membrane. It consists of four basic parts: the T7 promoter, RBS, Lpp-OmpA, and L-FABP. Lpp-OmpA is a sequence encoding a functional peptide that allows surface anchoring of its fused FABP. FABP can bind long chain fatty acids. By transforming this plasmid, the host bacteria can catch long chain fatty acids in liquid and carry it on its outer membrane.

Results

To confirm whether the pHisx6-Lpp-OmpA-FABP vector could successfully express the fusion protein, we used Western blot to analyze total bacterial proteins and cytomembrane proteins. The results showed that a protein with a size of predicted Lpp-OmpA-FABP fusion protein was clearly detected Follower E compared to the control group (Fig. 1A).Because Lpp-OmpA can anchor FABPs on the outer membrane, we determined whether FABP was present in the bacterial outer membrane. As we expected, FABP protein expression was steadily detected in outer membrane, but showed relatively low levels in cytoplasm (Fig. 1B). These results demonstrated that Lpp-OmpA could anchor FABP onto membrane; thus, pHisx6-Lpp-OmpA-FABP vector could work successfully, as we predicted.

Figure. 1. Western blot analysis of FABP protein expression in total bacteria (A) and cytomembrane (B). Con, empty pHisx6 vector; FABP, pHisx6-Lpp-OmpA-FABP overexpression plasmid; M, protein Molecular Weight Markers

To identify that the functional role of FABP in Follower E, the sample was tested using a kit designated for free fatty acids detection. The results showed that Follower E could efficiently bind fatty acids in culture medium when compared to the control, and the binding capacity of Follower E was higher in the medium with 400 μM fatty acids than that of 200 μM fatty acids. The result indicates that the FABP protein produced by Follower E could bind fatty acids as we designed.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 462
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 687