Team:Sydney Australia/Notebook


Week 1: 11 April - 17 April

  • Met up for the first time and were introduced to the project. Started to do some research on the project

Week 2: 18 April - 24 April

Wet Lab

  • Started to plan how we were going to approach the wet lab work: What reporter gene would we use to signify the presence of ethylene? How would we clone EtnR1 and EtnR2 into a plasmid to produce a functional construct?
  • Planned to split the wet lab team into subgroups for each arm of the wet lab work.
  • Started to do research on cell-based biosensors, cell-free paper-based biosensors, and other possible biosensor designs

Dry Lab

  • Brainstormed the role of modelling in iGEM and how it could assist in the experimental work.

Human Practices

  • Brainstormed possible outreach activities, including community science events, high school talks, Rotary Club talks, science activities at the university
  • Orion and Liam were nominated to volunteer at Investig8, an event organised by the University of Sydney’s Compass program, showcasing a few microbiology and molecular biology activities to high school students.

Week 3: 25 April - 1 May

Wet Lab

  • Began to brainstorm strategies for cloning work and the final biosensor construct.
  • Sing-young: Started to look into secondary structure prediction for EtnR1 and EtnR2, using PSIPRED.

Week 4: 2 May - 8 May

Wet Lab

  • Continued to research and gather ideas for the project, regarding biosensor design, ethylene metabolism in Mycobacterium strains, two-component regulatory systems in bacteria, secondary structure prediction of EtnR1 and EtnR2
Week 5: 9 May - 15 May

Wet Lab

  • Amanda and Orion: Designed gBlocks for EtnR1, EtnR2 and EtnP to be cloned into pBBRIMCS-2. For EtnR1 and EtnR2, gBlocks with the wild-type sequence and codon-optimised sequence (generated by the codon optimization tool from Integrated DNA Technologies) were designed.
  • Amanda and Orion: Designed primers for screening colonies containing EtnR1, EtnR2 and EtnP.

Dry Lab

  • Began weekly meetings with computational systems biology supervisor Ed.
  • Brief intro to mass action kinetics in biochemistry.

Week 6: 16 May - 22 May

Wet Lab

  • Ordered gBlocks and primers designed last week.

Dry Lab

  • Derived rate laws for enzyme kinetics reactions using Hill functions and mass action kinetics

Human Practices

  • Claudia: Created an iGEM Australia Facebook page for the four Australian teams, as a platform to communicate throughout the season.
  • Claudia: Contacted iGEM USYD 2015 and had a chat about their experiences.

Week 7: 23 May - 29 May

Wet Lab

  • Sing-young and Alma: Started designing primers to amplify and clone amilCP into pK18.
  • Sing-young and Alma: Started to research protocols for error-prone PCR, to mutate amilCP.
  • Contacted an academic, Dr Jacqueline Matthews, from the School of Life and Environmental Sciences who specialises in protein structure, function and engineering, to organise a meeting to discuss possible ways to study the structure of EtnR1 and EtnR2.

Dry Lab

  • Literature review of 2 component regulatory systems. Discovered a LOT of existing mathematical biology literature, but the focus generally seems to be on the abstract mathematical theory behind gene regulation and not applicable to lab/practical work.

Human Practices

  • Met iGEM USYD 2015, and got lots of advice about pressure points to watch out for throughout the season.

Week 8: 30 May - 5 June

Wet Lab

  • Met with Dr Matthews and discussed homology modelling and strategies to conjugate protein to a fluorophore, for a cell-free biosensor.
  • Amanda and Orion: Realised that the EtnR1 and EtnR2 gBlocks do not contain a ribosome binding site, which is required for their expression, so started thinking about designing a new set of gBlocks

Week 9: 6 June - 12 June

Wet Lab

  • Ordered new gBlocks containing ribosome binding sites.
  • Amanda and Orion: Decided to clone EtnR1 and EtnR2 into pET28c instead of pBBRIMCS-2, as it would allow the addition of an N-terminal hexahistidine tag for easy protein purification and downstream characterisation experiments.
  • Sing-young: Continued to work on structural modelling of EtnR1 and EtnR2.

Human Practices

  • Claudia, Liam, Wunna, Orion: Skype meeting with Antony Allen, from Avocados Australia.

Week 10: 13 June - 19 June

Wet Lab

  • Finalised cloning plan, from cloning the individual parts into expression plasmids, to the construction of the final biosensor.

Week 11: 20 June - 26 June

Wet Lab

  • Finalised cloning plan, from cloning the individual parts into expression plasmids, to the construction of the final biosensor.

Human Practices

  • Claudia: Made and distributed sponsorship prospectus.

Week 12: 27 June - 3 July

Wet Lab

  • Amanda and Orion: Started cloning EtnR1 and EtnR2 into pET28c.
  • Sing-young and Alma: Designed primers for amilCP error-prone PCR.

Dry Lab

  • Decided to branch off into software development to complement our biosensor. Initial ideas include a smartphone app to detect colour or a digital readout of the ethylene concentration.

Week 13: 4 July - 10 July

Wet Lab

  • Orion and Amanda: Continued cloning EtnR1 and EtnR2 into pET28c, and screening for positive colonies.
  • Sing-young and Alma: Performed a gradient PCR to determine the optimum annealing temperature for error-prone PCR of amilCP.
  • Sing-young and Alma: First attempt at error-prone PCR of amilCP.

Week 14: 11 July - 17 July

Wet Lab

  • Amanda and Orion: Sent positive colonies of EtnR1 and EtnR2 in pET28c for sequencing.
  • Sing-young and Alma: Ligated and transformed the first batch of mutant amilCP.

Dry Lab

  • Focused literature search away from gene regulation and towards finding parameters for transcription, translation and the ethylene signalling cascade.

Human Practices

  • Claudia: Attended the Amoeba Trade Display and pitched our project to potential sponsors.
  • Claudia: Spoke about the project at the Engineers Australia Women in Engineering Experience It! Student Conference.
  • Claudia: Spoke about the project on a student panel at the Explore Engineering Workshop.

Week 15: 18 July - 24 July

Wet Lab

  • Amanda and Orion: Continued cloning EtnR1 and EtnR2 into pET28c.
  • Amanda and Orion: Started cloning EtnP into pSB1C3 for parts submission.
  • Sing-young and Alma: Sequenced first batch of mutant amilCP, and started second round of amilCP error-prone PCR.

Dry Lab

  • Began forming final model using SimBiology. Quite a difficult learning curve!

Human Practices

  • Claudia: Email correspondence with potential sponsors.

Week 16: 25 July - 31 July

Wet Lab

Amanda, Orion and Claudia:

  • Confirmed correct sequences for both EtnR1 and EtnR2 in pET28c.
  • Confirmed correct sequence for EtnP in pSB1C3.
  • Started protein expression work: Transformed EtnR1 and EtnR2 into BL21(pLysS) cells which support IPTG induction of protein expression.

Sing-young and Alma:

  • Sequenced second batch of mutant amilCP, and started third round of amilCP error-prone PCR.

Dry Lab

  • First version of smartphone app produced. Discovered a bug that would detect white pixels as having a strong blue readout.

Human Practices

  • Claudia: Attended the Oz Harvest Think.Eat.Save Event at Martin Place.

Week 17: 1 August - 7 August

Wet Lab

  • Amanda and Orion: Continued testing expression of EtnR1 and EtnR2.
  • Sing-young and Alma: Sequenced third batch of mutant amilCP.
  • Sing-young and Alma: Carried out error-prone PCR with different MnCl2 concentrations to work out the optimal concentration for obtaining amilCP mutants.
  • Claudia: Carried out out Interlab experiments.

Dry Lab

  • First iteration of cellular model done in SimBiology. Dummy numbers have been assigned as rate constants.
  • Began coding of primer design app. Met with Dr Dale Hancock for a chat on primer requirements and research into what factors influence a primer.

Human Practices

  • Delivered first official presentation, to Biohacker, a biotechnology startup.

Week 18: 8 August - 14 August

Wet Lab

  • Amanda, Orion, Claudia: Could not confirm expression of EtnR1 and EtnR2 in BL21(pLysS) cells, so decided to transform EtnR1 and EtnR2 constructs into BL21(pGro7) cells, which express chaperonins that could assist in protein folding
  • Sing-young and Alma: Started fourth round of amilCP error-prone PCR.

Dry Lab

  • Transcription parameters: found Etr1-GST values for DNA binding activity. However, these are in units of radioactivity “counts” so they must be converted to a rate constant to be applicable to our model.
  • Primer design app: researched into an app that would potentially crawl multiple primer design software and collate all the results into a single platform.

Human Practices

  • Alma, Amanda, Claudia, Wunna, Liam: Volunteered at Science in the Swamp, entertaining and educating young children about science.
  • Claudia: Delivered a STEM talk at Gordon Library.

Week 19: 15 August - 21 August

Wet Lab

  • Amanda, Orion and Sing-young: Continued testing expression of EtnR1 and EtnR2 in BL21(pGro7) cells.
  • Alma: Sequenced fourth batch of amilCP mutants and started fifth round of amilCP error-prone PCR.
  • Claudia: Attempted the Interlab with protocol modifications.

Dry Lab

  • Found Ribosome binding site calculator that calculates ribosome affinity for a particular mRNA sequence. Possibly could use this as part of translation rate.

Human Practices

  • Hosted the JAMS (Joint Academic Microbiology Seminars) booth at the Australian Museum for Science Week.
  • Alma, Amanda, Wunna, Sholto: Manned a microbiology/molecular biology booth at the University of Sydney Open Day.

Week 20: 22 August - 28 August

Wet Lab

  • Amanda, Orion, Sing-young: Confirmed expression of EtnR1 and EtnR2 in BL21(pGro7) on SDS-PAGE gels, and sent the gels for mass spectrometry analysis for further confirmation.
  • Claudia: Started preparing a protocol and reagents for his-tag purification.
  • Alma: Continued fifth round of amilCP error-prone PCR.

Dry Lab

  • Discarded Ribosome binding site calculator in favour for ribosome speed across mRNA.
  • Transcription parameters: found promoter strength values for EtnR1, EtnR2 and the inducible promoter. For EtnR1 and 2 however, they must be converted from arbitrary RPU (relative promoter units) into Polymerases per second.
  • Found ethylene diffusion parameters.
  • First version of primer design app produced.

Human Practices

  • Claudia, Orion, Amanda, Alma, Liam: Skype meeting with Frank Bollen of Zespri Kiwifruits.

Week 21: 21 August - 4 September

Wet Lab

Amanda, Orion, Sing-young, Claudia:

  • Mass spectrometry results showed that EtnR1 and EtnR2 were successfully expressed.
  • Started purifying EtnR1 through Ni2+-affinity column chromatography.
  • Ordered detergent that needs to be used for preparing EtnR2 protein for purification, as it is a membrane protein.
  • Started to plan for protein characterisation experiments, including a gel-shift assay and pulldown assay to test EtnR1 binding to EtnP, and gas chromatography to test EtnR2 binding to ethylene.

Alma:

  • Sequenced fifth batch of amilCP mutants and started sixth round of amilCP error-prone PCR.

Claudia:

  • Attempted Interlab again with further protocol modifications.

Dry Lab

  • Found average mRNA degradation rates from Oxford iGEM 2015 wiki references.

Human Practices

  • Claudia, Orion: Visited Fresh Produce Group and had a tour of Flemington Markets.

Week 22: 5 September - 11 September

Wet Lab

  • Amanda, Orion, Sing-young and Claudia: EtnR1 purification yielded non-specific products, so troubleshooted purification through gradient elution.
  • Alma: Sequenced sixth batch of amilCP mutants, analysed sequences of mutants obtained so far.

Dry Lab

  • Created boundary and initial conditions for the scenarios to be modelled.
  • Looked into collaboration for 3 potential teams: Stony Brook, Hamburg and USNA Annapolis.

Week 23: 12 September - 18 September

Wet Lab

Amanda and Orion:

  • Continued to troubleshoot EtnR1 purification.
  • Finalised the cloning plan for assembling the final construct, and started cloning

Claudia and Sing-young:

  • Carried out gas chromatography to test EtnR2 binding to ethylene, using whole cells and cell extracts – Results did not show that EtnR2 was binding to ethylene; concluded that the assay was not suitable to test binding, as EtnR2 could be binding and releasing ethylene too quickly to be measured through gas chromatography

Alma:

  • Finished sequence analysis of amilCP mutants and decided on mutants to submit as parts.

Dry Lab

  • Contacted Hamburg iGEM2016 for parameters governing their two component regulatory system. Exchanged ideas for both our team’s modelling. Found that they were focused on an external model predicting the fluid flow behaviour in their micro device. However, their parameters were only available in arbitrary “docking score” units. Shouldn’t be a problem, we can still create a model with output in arbitrary units.
  • Contacted Hamburg IGEM2016 for smartphone app collaboration. Discovered that they have very similar requirements for their biosensor as ours and the smartphone app could potentially be used for their system as well.

Human Practices

  • Claudia: Delivered a talk at Ku-ring-gai Rotary Club.
  • Claudia: Attended an OzHarvest volunteer session.
  • Distributed survey on fresh food consumption and wastage.

Week 24: 19 September - 25 September

Wet Lab

Amanda, Orion, Sing-young and Claudia:

  • Carried out out a gel shift assay on semi-purified EtnR1, while continuing to troubleshoot purification.
  • Ordered Co2+ magnetic beads (Dynabeads®) for purification of EtnR1.
  • Continued to clone final construct.

Alma:

  • Grew up cultures of amilCP mutants to characterise their spectral properties.

Dry Lab

  • Diffusion model: Completed the explicit method for calculating local ethylene concentration at each node and timestep. Problems with graphical output in 3D plot but 2D plot seems to be working fine. Colours are not correlated with concentration, not sure why.
  • Completed Hamburg iGEM2016 smartphone app.

Week 25: 26 September - 2 October

Wet Lab

Amanda, Orion, Sing-young and Claudia:

  • Carried out purification of EtnR1 using Co2+ beads, which yielded a higher concentration of EtnR1 compared to the column purification.
  • Continued troubleshooting the protocol for the gel shift assay – Added more EtnR1; gel shift assay showed a shifted band!
  • Continued to clone final construct.

Alma:

  • First attempt at obtaining spectra for amilCP mutants – Could not obtain coloured soluble protein fractions.

Dry Lab

  • Diffusion model: Finished.

Human Practices

  • Attended an iGEM Australia meet-up at the University of New South Wales (UNSW).

Week 26: 3 October - 9 October

Wet Lab

  • Amanda, Orion, Sing-young and Claudia: Prepared to repeat gel shift assay with appropriate controls, to show that EtnR1 is binding to EtnP specifically
  • Amanda, Orion, Sing-young and Claudia: Continued cloning final constructs.
  • Alma: Designed primers and started cloning improved parts.

Dry Lab

  • Completed modelling the Hamburg iGEM2016 regulatory network, and wiki writing.
  • Completed the sensitivity analysis. Found that amilCP concentration was sensitive to only a fraction of the total number of parameters.

Human Practices

  • Claudia: Attended the ARC Training Advances in Biotechnology for Food and Medical Applications workshop.

Week 27: 10 October - 16 October

Wet Lab

  • Amanda, Orion, Sing-young and Claudia: EtnR2 purification was carried out using Co2+ magnetic beads (Dynabeads®).
  • Amanda, Orion, sing-young and Claudia: Finished assembling all plasmids for the final construct and transformed them into BL21(pGro7).
  • Alma: Refined protocol to obtain spectra for amilCP mutants and successfully obtained nice spectra!.
  • Alma and Sing-young: Finished cloning improved parts and sent for sequencing.

Dry Lab

  • Wiki writing and editing.

Human Practices

  • Hosted an iGEM Australia meet-up with Melbourne, UNSW and Macquarie teams, with each team presenting their projects and receiving feedback from the audience

Week 28: 17 October - 23 October

Wet Lab

  • Amanda, Orion, Sing-young and Claudia: Repeated gel shift assay with controls and concentrated EtnR1 (through dialysis) – Obtained a nice gel with a clear shifted band!
  • Amanda, Orion, Sing-young and Claudia: Tested the final construct by adding ethylene to the BL21(pGro7) cells induced for protein expression

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