Team:IIT Chicago/Results

iGEM IIT Chicago

Result

Result Weekly Summaries

Week of June 10, 2019
  • Primers were designed and ordered. Cyanobacteria was received. First attempts at PCR of pAM and PET.
Week of June 17, 2019:
  • Cyanobacteria subcultured. PET fragment completed and ready for Gibson assembly. New pCS primers were designed and ordered.
Week of June 24, 2019:
  • Ran PCR of fragments A-E and PET (control)
Week of July 1, 2019:
  • Ran PCR, gel, and amplification of fragments A, D’ and E.
Week of July 8, 2019:
  • Successful PCR confirmed by gel for fragments A and D’. PCR unsuccessful for fragment E.
15
Week of July 15, 2019:
  • Primers for fragment E redesigned and ordered. Preliminary conjugation protocol developed.
Week of July ,22 2019:
  • First successful PCR for fragments A and B. Amplification was run with taq and Q5 for both fragments.
Week of July ,29 2019:
  • Purification of fragments QA, QB and taq B. Gibson reaction of the primer A, B , pet-PET documented. Started liquid culture of pet_PET/3013.
    • Week of August 5,2019:
    • PCR and purification of fragments A and B with Q5. Gibson reaction ran and transformed.
    Week of August 12,2019:
    • PCR of Gibson reaction (also cultured and plated colonies). No successful Gibson reaction with the correct plasmid
    Week of August 19,2019:
    • Did mini preps of pet_PET and Gibson transformation
    Week of August 26,2019:
    • Re-ran gel of pET_PET to ensure colony growth. Made culture of pET_PET colonies grown from replica plate.
    Week of September 2,2019:
    • Made a liquid culture of pET_PET and did mini preps of four cultures (-6, -7, C, and C). Another Gibson reaction was set up and transformation was run. By the end of the week, no transformed colonies were observed.
    Week of September 9,2019:
    • Redid Gibson reaction with LBamp plates for control and performed transformation procedure. No growth was observed on Gibson plates once again. Made cultures of pCS_PET and pAM4788. Ran PCR for PET, A, and B. Also ran PCR for EE screening of -6, -7, C1, C2 from pET_PET and did protein purification.
      • Week of September 16,2019:
      • Purified fragments A, B and PET. Received a very low A260 value for PET frag, but OK A260 values for fragments A and B. Started new PCR of PET fragment to redo. Transformation of pAM4788 and Gibson into E. coli. Did high quality mini preps of pET_PET for assay.
        • Week of September 16,2019:
        • Made mini preps of four successful Gibson colonies and ran PCR screen.
        Week of October 7,2019
        • Redid Gibson and PCR of fragments A, B, and PET. Did Gibson extraction PCR using magic peg and had 11 colonies grow on the plate.
          • Week of October 14,2019
          • TFA assay was developed to prove that the degradation of PET nanoparticles tagged with fluorescent molecules could be tracked through fluorimetry.

          Final Words on Findings

          Green Ocean has had a long journey throughout the year in which we have advance greatly in our project. Even though the IGEM competition has already begun we are still working in the conjugation process of cyanobacteria, although we might not get to present conclusive results to all the IGEM team, we are not giving up with our project. We will continue to operate our project until our end state is achieved

          Citations

          • Borunda, Alejandra. “This Young Whale Died with 88 Pounds of Plastic in Its Stomach.” National Geographic, 22 Mar. 2019, https://www.nationalgeographic.com/environment/2019/03/whale-dies-88-pounds-plastic-philippines/.
          • Hornigold, Thomas, et al. “How Cyanobacteria Could Help Save the Planet.” Singularity Hub, 31 Jan. 2019, https://singularityhub.com/2018/06/04/how-cyanobacteria-could-help-save-the-planet/.
          • Long, Kat. “New Species of Bacteria Eats Plastic.” The Wall Street Journal, Dow Jones & Company, 10 Mar. 2016, https://www.wsj.com/articles/new-species-of-bacteria-eats-plastic-1457636401.
          • Spence, Edward, et al. “Membrane-Specific Targeting of Green Fluorescent Protein by the Tat Pathway in the Cyanobacterium Synechocystis PCC6803.” Molecular Microbiology, vol. 48, no. 6, Dec. 2003, pp. 1481–1489., doi:10.1046/j.1365-2958.2003.03519.x.
          • Whitaker, Hannah. “How the Plastic Bottle Went from Miracle Container to Hated Garbage.” National Geographic, 24 Aug. 2019, https://www.nationalgeographic.com/environment/2019/08/plastic-bottles/.
          • Wüstneck, Bernd. “In a First, Microplastics Found in Human Poop.” National Geographic, 23 Oct. 2018, https://www.nationalgeographic.com/environment/2018/10/news-plastics-microplastics-human-feces/.
          • Yoshida, Shosuke, et al. “A Bacterium That Degrades and Assimilates Poly(Ethylene Terephthalate).” Science, vol. 351, no. 6278, Oct. 2016, pp. 1196–1199., doi:10.1126/science.aad6359.

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