Difference between revisions of "Team:BGU ISRAEL/Design"

Protein Mutagenesis

Based on the structure of LC-Cutinase, we decided to use a rational mutagenesis approach, in which we introduce various mutations to the enzyme's sequence. The mutations were designed using the PROSS (Protein Repair One Stop Shop) algorithm, developed by the Fleishman Lab from the Weizmann Institute (Goldenzweig et al 2016).

The algorithm uses the following steps in selecting the mutations:

A sequence alignment was generated between the target wild-type sequence and other homologous sequences, from which several mutations were chosen.

Each mutant’s structure was compared to the wild-type structure, and the energy difference between them was calculated (ΔΔG_calc).

By computing the effects of each mutation separately, rather than in combination with others, design choices which included several ‘Potentially Stabilizing’ mutations (‘Potentially Stabilizing’ are mutations with ΔΔG_calc ≤ -0.45), were chosen, rather than designs that included several mutations, computed together and could include one or more non ‘Potentially Stabilizing’ mutations, but with a total ΔΔG_calc ≤ -0.45.

Here are a few examples of mutations present in our mutated variants:

Cells, Plasmids and Enzymes

E. coli strain MG1655 was used for cloning of the LC-Cutinase genes into the pACYC vector backbone. E. coli strain BL21 was used for protein expression. The wild type LC-Cutinase gene with the pelB leader sequence was obtained from the UC-Davis 2012 iGEM team (Part:BBa_K936013) and was amplified using primers containing restriction sites that match the pACYC backbone. The amplified gene was restricted with XhoI and BglII and ligated into pACYC using the T4 ligase enzyme.

After validating positive clones, activity tests were performed using a pNP-butyrate assay and PET degradation assays.

Microbial Fuel Cell

In order to build a self-sustaining system in which part of the energy from PET could be utilized to make clean energy, we decided to use microbial fuel cells (MFC).

We chose a bacterium that is proven to be electrochemically active (Timur et al 2007). Furthermore, it was essential that the bacterium would be able to form a biofilm over the electrode in attempt to improve the MFCs performance (Nielsen, L. 2012). P. putida KT2440 meets these requirements.

At first, we examined the possibility of adding an electron carrier to the fuel cell, as it was shown to be effective in enhancing the MFCs performance and was demonstrated using this species. We decided to use protocatechuic acid as it is part of the terephthalate metabolic pathway, which enables us to reduce the use of external reagents and to create a self-sustaining system. Protocatechuic acid had to meet four criteria:

1. The mediator redox couple should have a well-defined stoichiometry.
2. The mediator should be soluble and stable under the conditions present in the fuel cell.
3. The mediator’s formal potential should be adequate to the enzyme potential.
4. It should have a fast heterogeneous electrode exchange with the electrode and a fast homogenous electron exchange with the enzyme redox center or enzyme cofactor (Nuñez, M. 2005).

To examine some of those criteria regarding protocatechuic acid we plan to use cyclic voltammetry.