Notebook
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
|
|
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.
|
|
|
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). We digested these 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.
|
|
|
Wet Lab
Week 5 (31/7/17-4/7/17)
| |
Dry Lab
|
|
|
Wet Lab
Week 6 (7/8/17-11/8/17)
| |
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)
|
|
|
Wet Lab
Our plan was as follows:
- Make up new competent cells.
- Run a gel of PCR products.
- Transform cells using only 2ul HydEFG and new competent cells. Plate up these transormants.
- Make overnight liquid cultures of successful HydEFG cultures.
- Miniprep HydEFG from the above.
- Digest and run of gel the resultant extracted plasmid to check if worth sending for sequencing.
- 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.
|
Week 7 (14/8/17-18/8/17)
Week 8 (21/8/17-25/8/17)
| |
Dry Lab
|
Wet Lab
|
Week 9 (28/8/17-1/9/17)
| |
Dry Lab
|
|
|
Wet Lab
|
Week 10 (4/9/17-8/9/17)
| |
Dry Lab
|
|
|
Wet Lab
|
Week 11 (22/9/17-15/9/17)
| |
Dry Lab
|
|
|
Wet Lab
|
Week 12 (18/9/17-22/9/17)
| |
Dry Lab
|
|
|
Wet Lab
|
Week 13 (25/9/17-29/9/17)
| |
Dry Lab
|
|
|
Wet Lab
|
Week 14 (2/10/17-6/10/17)
| |
Dry Lab
|
|
|
Wet Lab
|
|
Week 15 (9/10/17-13/10/17)
| |
Dry Lab >
|
|
|
Wet Lab
|
Week 16 (16/10/17-20/10/17)
| |
Dry Lab
|
|
|
Wet Lab
|
Week 17 (23/10/17-27/10/17)
| |
Dry Lab
|
|
|
Wet Lab
|
Week 18 (30/10/17-3/10/17)
| |
Dry Lab
|
|
|
Wet Lab
|
