Team:Macquarie Australia/Notebook

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Dry Lab

  • The first week involved learning all about synthetic biology and what lies ahead in terms of the competition
  • Dr Louise Brown and explained all things iGEM including the biobrick system and 3A assembly
  • An overview of the chlorophyll biosynthesis pathway, photosystem II and hydrogenase activity presented by Professor Robert Willows since our project would focus on one component of this bigger picture
    • Our project being to use biosynthetic techniques to create the hydrogen producing hydrogenase
  • We started planning for creation of complete hydrogenase plasmid by combining the Ferredoxin/Ferredoxin reductase biobrick with the Hyd1 biobrick
    • From this we expect some preliminary production of hydrogen however, with the addition of maturation enzymes this production would be maximised.
  • Discussion on construction of the maturation plasmid in which HydE/HydF biobrick would be combined with HydG biobrick then the two larger constructs would be ligated together to create the complete plasmid coding for the total Hydrogenase molecular machine, fondly named Omega Ω
  • We created a “3-day plan” from 3A assembly through transformation and plating up cells to liquid media overnight cultures, miniprep kit extraction of plasmid and nanodrop concentration measurement, and finally electrophoresis gel screening of our newly made construct
  • Discussion on human outreach was started.
    • Began talking to the SDU iGEM team on collaboration
    • Discussed the creation of an iGEM Macquarie game
    • Started planning for attending synthetic biology conference and ACUR (Adelaide).

Dry Lab

  • India suggested the idea of a creating a game as her brother could help us out with that.
  • We got in contact with Joanne Jamie, an academic at Macquarie University, to gain more information about National Science Week and the university’s Orientation Day for our outreach possibilities.
    • She invited us to help out in the Chifley School Program (19th September) which we agreed would be better than the other two. We have a 1 hour time slot in which we can conduct wet and dry lab activities with gifted and talented high school science students.
  • The team was approached by Nebraska to fill out their survey
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Wet Lab

  • Checked lab supplies to ensure we had what we needed.
  • Became familiar with protocols we will be using.
  • Poured LB plates made up with each of the three antibiotics we will be using- Amp, Kan and CAM.
  • Conducted three transformations of DHα5 cells with DNA plasmids of Fer, Hyd1 and HydEF.
    • These were grown successfully on plates.
  • Dived into our first ligation attempt for Fer/Hyd following our “3-day plan”
    • Results = no growth on the plates.
    • This lead us to testing all our restriction enzymes on familiar plasmids, which gave us a chance to pour our own electrophoresis gel, load and run it.
    • It was great to have Mike Gibbs, Dominic Logel, Thi Huynh and Ed Moh supervising and sharing their expertise.

Dry Lab

  • Nebraska got back to us asking for collaboration: discussion of Australian policies in regards to synthetic biology.
    • In return we hope to get the survey results that we filled out to run our own questionnaire and compare Australian/Int’l response.
  • Had Skype call with SDU
  • India and her brother got back to the rest of the team with game ideas and responded to our feedback.
  • USyd contacted us to get in touch with the rest of the Australasian teams to organise a meetup.
    • We replied back saying we were interested.
  • ACUR Abstract submitted.
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Wet Lab

  • We reviewed the results from the restriction enzyme testing experiment conducted last week.
  • We interpreted electrophoresis gel results
    • XbaI did not perform well
    • SpeI appeared to be contaminated
    • We discovered that although 3.1 buffer is ideal for Pst when doing double digest it is ideal to use Cutsmart or 2.1.
    • New restriction enzymes are expected this week.
  • Conducted PCR of three backbones so we would have more stock available (CAM, Kan, Amp).
  • Mike re did our digest test of restriction enzymes. Ran electrophoresis gel of PCR’s and digests. Q5 didn’t work at all and KOD did not work for Kan.
    • Mike’s digests worked out.
    • The changes he used included Smart cut buffer used across all enzymes and digest extended to 90min.
    • The team re did the digest and re did Kan PCR again. Ran gel again. Digests look great but still no success with Kan PCR. Ed has suggested we try again with a lower temp for annealing as it’s possible our anneal temp was too close to the primer melt temp. He has suggested that the CAM and Amp pcr’s were not very successful either considering the main bands were weak and primer dimer bands strong.
  • We grew overnight subculture of three colonies from each of three plates- Fer, Hyd1, HydEF. The next day we extracted the plasmid using miniprep (Qiagen) followed by digestion using EcoRI as well.