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Revision as of 01:50, 3 October 2017

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Glossary

  • FNR - Ferredoxin NADP+ reductase.
  • Fer - Ferredoxin and Ferredoxin NADP+ reductase (FNR).
  • Hyd- Hyd 1 coding for the enzymatic part of the Hydrogenase molecular machine.
  • Fer/Hyd- Fer and Hyd as described above in a new biobrick construct.
  • HydEF- Hyd E and Hyd F, coding two of the three maturation enzymes.
  • HydEFG- Hyd E, Hyd F and Hyd G, coding all three maturation enzymes in a new biobrick construct.
  • Fer/Hyd/EFG or Omega Ω - plasmid with all the necessary genes coding the total Hydrogenase molecular machine. Ferredoxin, Ferredoxin NADP+ reductase, Hyd 1, Hyd E, Hyd F and Hyd G all in a new biobrick construct

    • Week 1 (3/7/17-7/7/17)

    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).


    Week 2 (10/7/17-14/7/17)

    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.

    Week 3 (17/7/17-21/7/17)

    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.

    Week 4 (24/7/17-28/7/17)

    Dry Lab

    • Unofficial team meeting took place as some team members still absent.
      • To Do List was discussed.
    • There was a meet-up with Ari, Steve, Indi, Louise and Catherine (Pop Up Incubator) where we consult in a game they are designing.
    • Discussion around potential collaboration opportunities with CSIRO.
    • Game avatar design finalised and comments shared amongst the team.
    • Abstract edited for SynBio by India and given to Louise to hand in for registration.
    • USyd approached to form Australasian Meeting for iGEM-they paid, we dont want to. Need to find out if UMelb and Auckland (other invitees) have paid-if not allows us leverage.

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    Wet Lab

    • We were ready to re-attempt the three day plan which starts with 3A assembly ligation of the lac-ferredoxin- FNP with the lac-hydrogenase.
      • As these were both in CAM backbones, they were digested then ligated into Amp backbone.
      • We used new incubation settings for ligation which ramped up the temperature based on advice from Rob.
      • Transformed cells were grown on plates overnight.
        • We were excited to achieve one colony of our Fer/Hyd plasmid cells however with very low numbers even on the puC19 positive control plates the competency of the cells was in question.
        • At the same time we ligated the newly arrived biobricks- GUN4, HydG and Double terminator into CAM backbones.
      • These were also transformed into cells and successfully grown overnight.
      • Subcultures of these as well as the one Fer/Hyd colony were grown in liquid media ready for miniprep.
    • PCR (both KOD and Q5) was attempted again on all three backbones and the gel showed disappointing results.
      • Having discussed this with Rob it was decided that our templates were a poor choice (iGEM linear backbones).
      • Our next attempt to amplify the backbones- CAM, Amp and Kan utilised the RFP/antibiotic resistant backbone plasmids from previous iGEM members.
    • While running PCR products on gels we had the opportunity to tweak our NEB 1kb ladder.
      • The ladder used was a 1:10 dilution of the original now with loading dye added to the mix so we could confidently just drop 2uL in a well and see a great result every time.
    • In an attempt to test the competency of the cells we’d been using we challenged Thi’s cells against Rob’s cells (all DH5⍺).
      • We transformed each with our Fer/Hyd ligation and with puC19 +ve control.
      • As we plated up the cells someone noticed that our heat block temp was not set correctly so effectively our transformants had never been heat shocked.
        • Given these probably won’t work we re-did the transformation.
      • The next day we found those that weren’t heat shocked did grow very few cells for the puC19 +ve control and none for Fer/Hyd.
      • Rob’s cells showed more colonies. 3 colonies for Fer/Hyd.
    • We had also grow one more colony on a plate from spin down of the previously transformed Fer/Hyd cells which had be held in reserve in the fridge.
    • Sent off for sequencing were- lacHydG, Double terminator, Gun4 and our Fer/Hyd construct.
    • That was the last week of what was fondly known as bootcamp. Now that the vacation period is over we will be challenged with university classes and other challenges to our lab time.

    Dry Lab

    • Some wiki layout ideas were drawn up by Winonah.
    • Our promotional sponsorship brochure was drafted and so was a powerpoint presentation, which was far less professional and more suited to a personal viewing.
    • A logo competition was started (set up by Ari) to choose which logo would best represent our team.
      • The designs are still being created to be voted on at a later date.
    • Wiki team profiles were drafted and needed to become more succinct as they seemed bulky on the wiki.
    • Project title ideas were considered
    • Our abstract needed changing to include some dry lab activities.
    • GoGreen collaboration was introduced as a possible collaboration.
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    Wet Lab

    • Sequencing results came back and unfortunately our Fer/Hyd plasmid was lacking Hyd 1.
      • This was unexpected.
    • We miniprep extracted Fer/Hyd plasmid from the three new colonies which grew from Rob’s cells, as well as the one colony we achieved from previous cells spun down.
      • Once run on gel, two from Rob’s cells (A and B) looked promising so these have been sent off for sequencing.
    • A new ligation was made with HydEFG and Thi’s batch of cells transformed with this new construct.
      • Unfortunately, nothing grew.
      • With 5ul of HydEFG plasmid still available we decided we should reduce our transformations to use only 2ul.
      • This would leave us enough to digest and run on a gel next week.
      • The plan was to make up new competent cells as it appeared the cells we are using were not as competent as expected.
      • Keen to keep going we proceeded with commercially available competent cells of our next HydEFG transformation attempt.
    • Running a gel showed that the latest attempt to PCR amplify the three backbones- Amp, CAM, Kan, was not successful.
      • Another attempt has been made to achieve this using different templates.
      • We plan to run these on a gel next week.

    Dry Lab

    • Game design was discussed in regards to background animation and sounds. The main character was finalised.
    • Our promotional brochure was completed and the powerpoint presentation was abandoned in favour of it.
    • The team decided to go ahead with the GoGreen collaboration.
    • Sponsorship was finalised. Emails went out to new potential sponsors and contact was made with previous sponsors. Other fundraisers discussed included an Indiegogo page (linked to Adrianna) and potentially Grill’d “Local Matters.” (linked to Ari). Paul Jaschke advised of Twist Bioscience sponsorship opportunity, so Ali started working on an application for it (closed 25th August).
    • Logo design contest closed and decisions progressed towards a winner (closed Monday 14th August) .
    • Official announcement that India will be presenting at ACUR Adelaide. Funding paperwork was underway and due on the 16th August. Evidence of flights/accommodation was due on 21st August so India and Ali worked on this. Ali sent an email to the Office of the PVC with questions.
    • Received an update from USyd about their project and forwarded email to Thi who thinks we may be able to collaborate (we have a lot of E.coli strain that they are using). Emailed back with our project.
    • Steven has uploaded his hypothesis and survey questions for review
    • On Tuesday Ari, Steven, and India had a meeting with the Dr Egg team to consult on the scientific themes incorporated into their puzzle games and brainstormed ideas. It was discussed that one of us will be at a table with a game developer and 4-5 kids for 30 minutes at a time to explain the science background of each theme and have the kids develop game ideas. It was also discussed that we would do a 10-15 minute presentation at the beginning of the event to give a basic introduction to synthetic biology and our specific iGEM project. (Event will be on 18th of August)
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    Wet Lab

    Our plan was as follows:
    1. Make up new competent cells.
    2. Run a gel of PCR products.
    3. Transform cells using only 2ul HydEFG and new competent cells. Plate up these transormants.
    4. Make overnight liquid cultures of successful HydEFG cultures.
    5. Miniprep HydEFG from the above.
    6. Digest and run of gel the resultant extracted plasmid to check if worth sending for sequencing.
    7. If result from FER/Hyd A and B sequencing is unsuccessful then a new ligation of Fer/Hyd should be attempted. Look at what might need tweaking.
    • Instead of making up new competent cells, we used commercial competent cells which proved successful as we were able to grow HydEFG transformants.
      • Plasmids from these were extracted via miniprep.
      • They were digested and run on a gel.
      • The gel results were not as expected _____. Because of this, new colonies should be screened.
    • The sequencing came back for Fer/Hyd A and B. Both were as confirmed.
    • The glycerol stocks were then used to prep overnight cultures which were induced and tested for their ability to produce hydrogen.
    • The Clark electrode was used to measure the H2 production and recordings went off the scale. This was great news. We are looking into the best ways to present this data.
    • Unfortunately we still have not had any success with PCR amplification of backbones.
    • HydEFG were sent to be sequenced and other cultures were grown as well as their glycerol stock.

    • Organisation of ACUR conference.
      • ACUR funding submitted, India and Ali are preparing slideshow presentation.
    • Indiegogo online fundraiser set up by Adrianna.
    • Ali, Adrianna and Winonah attended a meeting with a potential key sponsor (Bioplatforms). They discussed all aspects of the project from human practices through to the wet lab.
      • Currently waiting to hear back from the company.
    • Dr Egg Digital: Team members Ari, Steven and India collaborated with a team of game developers and artists from Dr Egg Digital as science consultants for a game testing and puzzle planning event.
      • Led by Dr Catherine Fargher, the concept of the videogame is based from her successful theatre show Dr Egg and the Man with No Ear and chapter book publication.
      • A class of 8-12 year olds from the Queenwood School Catalyst Program were audience to an age appropriate lecture led by the Macquarie iGEM team on the basics of synthetic biology and its applications, and the possible future prospects of our project in renewable energy.
        • The lecture spurred the creativity of the children who then brainstormed game puzzles and character scripts for the Dr Egg Digital game that is being designed to incorporate the national school science curriculum for grades 3-8.
        • The resulting ideas from this collaboration will be incorporated into this educational videogame that is set to be released in February 2018.
      • The event was part of Australia’s National Science Week 2017, with our project aptly relating to this year’s theme being ‘Future Earth’.
      • Impressed by how this event engaged their students, Queenwood School shared information of our iGEM team and the community outreach we have participated in on their social media channels and their school publications.
        • “Thanks for your AWESOME work yesterday! Lindy the main teacher from Queenwood said she has never seen them so engaged.” – Dr Catherine Fargher (Dr Egg Digital)
        • “Dear Dr Walsh, thank you for letting me come to the Dr Egg excursion. It was really fun, it was possibly one of the best days of my life!!!! I loved inventing the game about Vivi. P.S. glowing Cats. P.P.S cat DNA with glowing jellyfish DNA with narwhal horn DNA = cat-a-corn." – Siana (Queenwood School student) Dr Egg Digital Website
    • Open Day: Team member India co-presented a lecture with the head of department for Chemical and Biomolecular Sciences, Professor Alison Rodger, at Macquarie University’s Open Day.
      • The lecture introduced the basic concepts behind synthetic biology, what to study to enter a career in this field, and introduced the iGEM competition and our project to prospective new students.
      • A similar talk was completed by Ali, with Jenny Donald, to incoming BSc (Human Bio) and BMedScs students, on what iGEM is and Macquarie’s history in the competition.
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    Wet Lab

    • This week had a major focus on completing the Interlab study (Adrianna, Winonah, Jocelyn, Ali, Adrianna, Indi, Ed and Thi were involved)
      • Transformations and overnight cultures were completed in time for the big day of data collection on Thursday which included the Interlab fluorescence measurement of plates.
      • We started early on the interlab task, first we made fresh cell cultures from the cells that had been incubating over night. Then we started with the absorbance measurements of _____. We then made our time 0 samples, put the new cell cultures in the incubating shaker and left for 2 hours.
        • During the 2 hour period the serial dilution plate was set up ____
      • Towards the end of interlab day, Team Member numbers were dwindling so Ed and Thi assisted with some pipetting to ensure we would be able to obtain spectrometer readings that day.
    • At the same time, a backbone swap to CAM for Fer/Hyd took place followed by transformation and plating up.
    • Hydrogen production data collection by Clark electrode continued.
    • HydEFG was digested (single and double digests performed on 7 separate colonies) and screened on gel (#V, 45 mins). Meanwhile we await sequencing result.
      • As the gel was running a team member messaged the group saying that unfortunately the protocol used was not an updated version…
      • After visualising the completed gel, it appeared the digests were only successful for 3/7 samples?
    • No more backbone PCR amplifications took place this week.

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    Week 14 (2/10/17-6/10/17)

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