Experiments Overview

Our experiment aims to engineer a chassis to degrade micro-plastics in an efficient and accessible manner in the natural environment. We utilized PCR, recombination and transformation to construct plasmid for E.coli to express the PETase. The mutated PETase and signal peptide sequence were inserted during this process for enhancing efficiency and out-membrane PETase expression. Then, we changed our chassis to Cyanobacteria due to its self-sufficient characteristic. Last, the modified Cyanobacteria was co-cultivating with Rhodococcus to further break down the monomer initially broken down by PETase.

Experiments Process

1. Initial Construction:

  1. Plasmid Extraction: extract the plasmid with our wanted segments (PETase) from iGEM distribution kit
  2. PCR of Targeted Segment: acquire and amplify the segments that we want from the plasmid
  3. PCR Product Purification: get rid of unwanted DNA, like the original plasmid, and other unwanted substances from PCR product
  4. Assembly: assemble PETase segment with constructed shuttle vector backbone
  5. Transformation into DH5α: transform the constructed plasmid into competent DH5α E.coli cells
  6. Colony PCR: to see whether the plasmid is transformed into E.coli or not
  7. Sequencing: send samples to sequencing company and get the exact sequence of constructed plasmid to check whether construction was correct

2. Mutation construction:

  1. Normal PCR: acquire PCR product with mutated PETase
  2. Assembly: assemble PCR product and signal peptide gene with linearized pET28a plasmid
  3. Transformation into DH5α and Further Transformation into BL21(DE3): transform the constructed plasmid into competent DH5α E.coli cells; further transform plasmid into BL21(DE3) for protein expression

3. E.coli Expression

  1. Protein Induction: cultivate the positive single colonies of BL21(DE3), add IPTG to induce the start of transcription and the expression of PETase
  2. SDS-PAGE: check the size of the proteins that were expressed before and after induction
  3. Enzyme Assay:
    1. p-nitrophenyl butyrate Degradation Assay: detect PETase’s activity to check the degradation efficiency
    2. Scanning Electron Microscope: to observe the degrading level of microplastic

4. Synechococcus elongatus PCC 7942, Co-culture, and Final Justification

  1. Natural Transformation into Synechococcus elongatus PCC 7942: transform the plasmid constructed from BL21(DE3) into Syn 7942
  2. Streaking Cultivation: culture positive monocultures in preparation for colony PCR
  3. Colony PCR: to check whether the plasmid has been successfully transformed into Syn 7942 or not
  4. Enzyme Assay:
    1. P-nitrophenyl butyrate Degradation Assay: detect PETase’s activity in Syn 7942 to check the degradation efficiency
    2. High Performance Liquid Chromatography (HPLC) check of E. coli product: to get a chromatogram (chemical composition) showing the products that the proteins have produced, and justify that the product consists of TPA
  5. Co-cultivation: cultivate Rhodococcus jostii and modified Syn 7942 together to allow for further breakdown of TPA

Protocols