Experiments

Experiments Overview

This experiments page includes the protocols we used in our wet lab experiments, and a breakdown of our experiment steps, from acquiring the targeted segment to constructing plasmid in E. coli to transforming the constructed plasmid into cyanobacteria to finally checking the production of the terpenes through GC-MS.

Process ///

• Plasmid extraction: extract the plasmid with our wanted segments (terpene synthase genes) from product we bought
• PCR of targeted segment: acquire and amplify the segments that we want from the plasmid
• PCR product purification: get rid of unwanted DNA, like the original plasmid, and other unwanted substances from PCR product
• Assembly: connect the targeted segments to the shuttle vector backbone
• Transformation into DH5α: transform the plasmid constructed into competent DH5α E. coli cells
• Colony PCR: to see whether the plasmid is transformed into the E. coli or not, and get the rough size of the transformed targeted segment
• Plasmid extraction: extract the plasmids from single colonies with positive colony PCR, can be used either for sequencing or for later transformations
• Sequencing: send samples to sequencing company and get the exact sequence of constructed plasmid
• Protein check:
  • Normal PCR: acquire PCR product with terpene synthase genes
  • Assembly: assemble PCR product with linearized pET28a plasmid
  • Transformation into DH5α: transform the plasmid constructed into competent DH5α E. coli cells
  • Colony PCR:to see whether the plasmid is transformed into the E. coli or not, and get the rough size of the transformed targeted segment
  • Plasmid extraction: extract the plasmid for further transformation into BL21(DE3)
  • Transformation into E. coli BL21(DE3):transform previously constructed plasmid into BL21(DE3)
  • Colony PCR: to see whether the plasmid is transformed into the E. coli or not
  • Protein induction: cultivate the positive single colonies, add IPTG to induce the start of transcription, and the expression of protein
  • SDS-PAGE: check the size of the proteins that were expressed before and after induction
• GC-MS check of E. coli product: to get a chromatogram (chemical composition) showing the products that the proteins have produced
• Transformation into Synechocystis sp. PCC 6803: transform the plasmid constructed in DH5α into Syn. PCC 6803
• Streaking cultivation: culture positive monocultures and induce them so production of terpenes can be initiated
• Colony PCR: to check whether the plasmid has been transformed correctly into Syn. PCC 6803 or not
• Cultivate and induce monocultures: culture monocultures that have been transformed successfully and induce them so production of terpenes can be initiated
• GC-MS check: to see the products that the Syn. PCC 6803 has produced through chromatogram

Protocols ///