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Revision as of 17:09, 17 October 2016

PlastiCure

Design

Rational Mutagenesis

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 matching the pACYC backbone. The amplified gene was restricted with XhoI and BglII and ligated into pACYC using the T4 ligase enzyme.

After cloning activity test were performed using a pNP-Butyrate assay and PET degradation assays.

Protein Mutagenesis

Based on the LC-Cutinase structure, a rational mutagenesis approach was chosen for our project. In this approach, various mutations were designed using the PROSS (Protein Repair One Stop Shop) algorithm that was developed by the Fleishman lab from the Weizmann Institute (Goldenzweig et al 2016). 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 (ΔΔGcalc).

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 ΔΔGcalc ≤ -0.45 Rosetta energy units(R.e.u.)), 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 ΔΔGcalc ≤ -0.45.

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

A249P - Proline adds to the rigidity of the protein structure.
M259F - A ‘hole’ inside the protein core could be destabilizing to the protein structure. Phenylalanine with a benzyl side group (hydrophobic) is used to ‘fill’ the hole, thus making the protein more stable.
S257A - Polar side groups, such as a hydroxyl in Serine, with no hydrogen bonds, are destabilizing as the protein core should be hydrophobic. A mutation changing Serine to Alanine should increase stability.

Metabolic Pathway Engineering

Two inserts were designed to transform the genes necessary for the terephthalate metabolic pathway to the Pseudomonas putida bacterium. Two vectors for cloning were chosen and ordered from the Standard European Vector Architecture’s (SEVA) collection. The 2 vectors chosen are pSEVA224 and pSEVA434. We have chosen the pSEVA plasmids because they have been previously used in the P. putida KT2440 strain by the D. Taufik group from the Weizman Institute, who we acquired the bacteria from.

Two inserts were designed –

  1. Containing 4 genes from the same strain of C. testosteroni for the degradation of Terephthalic acid to Protocatechuic acid – tphA1, tphA2, tphA3, tphB.
  2. Containing 3 genes from C. testosteroni strain KF-1 for the transport of Terephthalic acid from the periplasmic space into the cytoplasm – tphC, tctA, tctB.

The genes involved in the Terephthalate degradation pathway and transporter were acquired from the 2012 Darmstadt iGEM team (Link).

Prior to each gene, we added the broad-host G10L ribosome binding site, derived from bacteriophage T7 for increased expression. To provide us with an option to excise a specific sequence from the designed insert if needed, we designed a restriction cut site between every element (gene or RBS). The cut site had to be unique to avoid cutting the genes or the vector backbone.

Flanking each insert were sequences homologous to the desired target vector (224 or 434) for cloning using the NEBuilder® HiFi DNA Assembly Cloning Kit. Furthermore, to provide an alternative cloning strategy, the flanking sequences contained restriction cut sites for cloning into the MCS using restriction digest and ligation.

Our design for efficient degradation of PET required the symbiosis our our two chosen bacteria - E. coli and P. putida, so we had devised an apparatus to enable their mutual growth without contact. Our chosen approach utilized a dialysis membrane with a cut-off of 12-14kDa (lower then LC-Cutinase's molecular mass) to enable the diffusion of the PET degradation products outside the bag for utilization by P. putida, while enabling a large concentration of LC-Cutinase inside the bag for efficient degradation of PET. Shredded PET was added to the dialysis bag and a magnetic stirrer was used to prevent precipitation.

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 takes part in the terephthalate metabolic pathway, enabling 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.

Address:

Ben-Gurion University of the Negev
Ben Gurion 1, Beer Sheva 8410501, Israel

Mail: igembgu2016@gmail.com

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