Team:BABS UNSW Australia/Notebook

Notebook

    A brief summary of our trials, tribulations and fleeting moments of joy.

    Late November - Early December 2014

      Following a lecture recruitment hustle by Rob, team members applied and were selected for the iGEM team. We all started reading up on synthetic biology and dreaming potential projects ranging from far-fetched to science fiction to milk.

    17-19 February 2015

      The team attended a three day bootcamp run by Rob, Daniel and Chris. We learned the basics of molecular biology techniques for the iGEM competition - cloning, enzyme digests, ligations and transformations. We participated in many sessions (some lead by team members) about other aspects of the competition - wiki design, primer design, actual design design. We also started presenting and discussing project ideas, and brainstorming ways to make each of them better.

    2-6 March 2015

      The uni semester starts, and we had our first meeting on the first day. We discussed communication strategies (online labbooks, OneNote vs Evernote) and role allocation. Most importantly, we started seriously talking about various projects ideas and their respective advantages and disadvantages.
      Projects discussed included:
      • recombinant dehalogenases
      • dynamic gene editing
      • type 3 secretion systems for protein delivery
      • biophotovoltaic cells
      • synthetic endosymbiotic relationships for therapeutic use (endosynbio!)

    9-13 March 2015

      Despite our 8am Monday morning meeting, we enthusiastically discussed a narrowed down list of projects, and set down pros and cons for each. After some discussion and a preferential vote, we settled on Endosynbio! Despite lying on the sci-fi end of the spectrum, we found some seemingly achievable goals to achieve with our project and decided it was worth the risk. The three parts as we decided were:
      • invasin/listeriolysin
      • colA/minC control system
      • xenophagy
      We also started the iGEM registration process and that was exciting.

    16-20 March 2015

      At this Monday morning meeting, we discussed a wide range of topics. We started getting organised in terms of funding, outreach, wiki, team roles and labwork. We were all still spending a lot of time reading up and dreaming up project and outreach ideas.
      We had a separate design meeting (Kris, Mac, Daniel) where several ideas, including the DNA rainbow serpent, were borne (and rapidly buried).
      Not satisfied with our one-hour meetings, the team (minus advisors) planned to meet all day on saturday. It was surprisingly fun, productive, and everyone left feeling optimistic.

    23-27 March 2015

      Monday morning meeting: more project discussion. What symbionts/host cell lines should we use? Synechocystis seemed to be the favourite. We also decided on the importance of a very effective kill switch.

    30 March-3 April 2015

      Another early Monday morning meeting. Prior art searches showed significant similarity of our planned project to two previous iGEM projects (Warsaw 2009-2010). The main concern of this meeting was finding ways to differentiate our project from those, and make it novel. Ideas included a focus on population control within the cell and making the host reliant on the endosymbiont.
      We finished writing our first grant application, for a grant offered by NSW Trade & Innovation.
      We investigated flights to Boston in September. Australia is very far from the rest of civilisation, so this was important.

    13-17 April 2015

      A difficult week for the team, as we dealt with the loss of one our team members. The project took a hiatus as we mourned Ben. Words cannot express the shock and deep sadness we all felt. Though it seems like a strange forum to discuss such a tragic event, to gloss over it would be impossible. His absence was deeply felt. Several team members attended his funeral this week, and this further emphasised the tragedy of his passing. He was surrounded by loving family and friends. His ambition and passion for science and helping others has driven and informed the rest of our project. Rest in peace, Ben. Words on a wiki cannot do you justice, but I feel like you would appreciate it.

    20-24 April 2015

      We had our weekly Monday morning meeting, and a separate wiki and kill switch design meeting.
      We started lab work: making media (LB and SOC) and pouring agar plates.
      The distribution kit arrived!

    27 April-May 1 2015

      This was an assessment-heavy week for all of us, so not much progress was made. However, we managed to write and distribute our first monthly newsletter.
      Wetlab:
      This week consisted of familiarising ourselves with the lab and with using the iGEM distribution kit. Four BioBrick parts related to our project were resuspended and transformed into E. coli. All successful transformants were miniprepped to isolate their plasmid DNA, which was then quantified via NanoDrop/Qubit and visualised via PCR and electrophoresis. Stocks of LB and LB agar were prepared as well as TAE buffer for future gels. Our culture of Synechocystis has also been incubating this week, with significant green growth now evident.

    May 4-8

      Many things starting to progress on the outreach front - collaboration discussions with the BIOMOD team, Izzy working on SmartSparrow programming and getting feedback from high school teacher surveys, and the conception of a UNSW-hosted Aussie iGEM meet up. We were all starting to feel a bit lost with our project due to the lack of clear goals, and many problems becoming apparent.
      For example:
      • Not different enough from past projects.
      • Synechocystis issues such as long doubling time, transformation and lack of expression tools. Furthermore we lack a strong validation for why we want to use Synechocystis.
      • It is unclear what we can reasonably achieve with the project as in its current form. If Synecho fails or is too slow we won't have much.
      This is when we started to consider using Lactococcus and E. coli symbionts in addition to Synechocystis. There was too much to talk about, so we scheduled in another mega-meeting. We spent several hours, and came up with many ideas that formed the final project, such as the pHlow system and the idea of trialling several symbionts in several different mammalian cell lines in several conditions/concentrations.

    May 11-15

      During our Monday morning meeting we discussed the results of our mega-meeting. The key goals of our project are now:
      1. invasion: Invasin and LLO ph induced expression. Improvement on previous systems as it responds to the environment and does not produce LLO unless it needs it. This system has been named phLOW. It will work in partner with the cre/lox safety switch.
      2. Safety/Kill switch: Lox sites flank the invasion genes with Cre downstream and under the influence of the same promoters. This should result in the invasion genes excision and subsequent degradation after they have been expressed to invade a cell. We may need to play around with slowing down the transcription via rare codon elements or pseudoknots. If possible we aim to make the kill switch be positively selected for, one suggestion is using an auxotrophic strain and amino acid synthesis on the plasmid. Another is transferring an essential gene from the chromosome to the plasmid.
      3. Population control: AHL mediated population density sensor that alters gene expression to produce an antimicrobial peptide. This would result in a dynamic, oscillating population density.

Inspiration

You can see what others teams have done to organize their notes: