Team:Sydney Australia/Future

Future Work

Our project is largely driven by our bioinformatics and modelling work. Future work could involve testing our methods and parts in the lab, and thus determine if natural transformation is possible through this system. Some questions to answer could include:

  • Have we successfully inserted our gene clusters into the E. coli genome?
    • This could involve modelling the uptake rates via recombineering, or even sequencing transformed cells to determine if the clusters are being inserted at the expected location.
  • Are our natural transformation genes being transcribed as expected?
    • The mRNA of the cells could be extracted and analysed, providing transcriptomic data to analyse for the presence of the A. baylyi transcripts.
  • Are our natural transformation genes being translated as expected?
    • Proteomics analysis could be employed here as well, or western blots to determine the presence of certain proteins. The western blots could be run against a control colony to observe any differences.
  • Are our natural transformation genes functioning as expected - have they built the type IV pilus DNA uptake machine?
    • We hypothesise that the structure would only begin to take shape after all the cluster have been inserted.
  • How efficient are our Free Coli cells taking up and integrating foreign DNA?
    • This should be tested with iGEM plasmid pSB1C3 after each cluster insertion. This not only provides insight into the key genes involved with natural transformation, but it can also guide the design cycle if there is a sudden drop in transformation after a cluster insertion and that needs to be investigated.

On top of that, there is a design element of the project that could be optimised if it was informed by lab data:

  • Minimising antibiotic resistance in Free coli
    • One thing to note about the Babushka Block method is that the Free coli cells at the end will still have resistance to certain substances. With this current design, it will have the qacE gene after the final insertion, so it will be resistant to bactericidal quaternary ammonium compounds.
    • As described in Selectable Marker Design, switching Cluster 7 and 8 would allow the last selectable marker to be not related to antibiotics, but whether this is possible is only known through testing it.
    • We were also experimenting with the idea of using negative selection to create a selectable marker free end product. tetA is known to negatively select against nickel chloride (Ryu et al., 2017), so if the penultimate cluster had the tetA gene, the last cluster does not even need any selectable marker. This could be tested in the lab if this is possible, and then the selectable marker in Cluster 7 could be replaced with tetA.

    In regard to future work in the area of human practices and integrating human practices into the implementation of Free Coli, our team has proposed the following:

    • From our review of current Australian and international gene technology legislation, regulation and governance paradigms, our team identified several areas ripe for further community consultation and policy development. As part of our proposed future work, we believe there is a need to improve the proactive education on gene technology regulation for the wider community, as well as consult them on any potential legislative paradigm changes, such as a shift to considering benefits as well as risks as opposed to the status quo, which does not factor potential benefits to the community and/or environment into regulatory decision making.
    • The review also raised the issue of policy reform to better cater gene technology regulation to emerging 'DIY' synthetic biologists - a new trend that represents both potential and risk. Further work proposed by our team includes the development of a Gene Technology Regulation and Community Safety Educational Toolkit to educate DIY synthetic biologists on gene technology regulation and safety requirements in Australia. This toolkit could also be used as part of collaboration with future iGEM teams to adapt the toolkit to a multitude of legal jurisdictions around the world.


    Ryu, Y. S., Chandran, S. P., Kim, K., & Lee, S. K. (2017). Oligo- and dsDNA-mediated genome editing using a tetA dual selection system in Escherichia coli. PLOS ONE, 12(7), e0181501.