From 2011.igem.org

Localization

To improve the efficiency of conversion of acyl-ACPs through a two-step pathway to alkane production, we have undertaken optimization through localization of the two relevant proteins. Decreasing the distance intermediates must travel between Acyl-ACP Reductase (AAR) and Aldehyde Decarbonylase (ADC) is intended to improve overall alkane production.

We also have adapted Biobrick parts for use in alkane production from the 2010 iGEM Slovenia zinc finger violacein biosysnthesis project. By colocalizing the individual violacein producing enzymes in a five-step pathway, they were able to increase their pigment production by ~6-fold.

Direct Fusion

Our first approach was direct fusion of two proteins using a glycine-serine linker. We chose to construct both configurations of AAR to ADC linkage to test the effects of fusion by N and C terminus regions on enzyme performance, resulting in both an AAR to ADC and an ADC to AAR fusion.

Zinc Finger Fusion

In an attempt to enhance our localization technique beyond the simple protein fusion method, we chose to integrate the use of DNA localization via zinc finger proteins to co-localize ADC and AAR to increase system flux. Zinc fingers are small DNA binding domains, consisting of around 30 amino acid residues folded around a single zinc cation. They function as transcriptional factors within eukaryotes, regulating endogenous gene expression by binding to specific sequences along DNA. Last year's Slovenia iGEM team characterized 6 zinc fingers and created a DNA program containing specific binding sites for each zinc finger. Our goal was to fuse AAR and ADC to their own respective zinc finger, such that when co-expressed with the DNA program, the fusion proteins would each bind to their specific target sequence along the DNA, thus being held in close proximity, so that alkane production could be carried out more efficiently.