Future Improvements

Expanding the repertoire of expression outputs

The use of three fluorescent proteins was a logical starting point for the exemplification of our system and proof-of-concept experiments. Nevertheless, the system design allows effortless expansion of the possible expression outputs.

On one hand, the number of expressed proteins could be increased as desired, in combination with the expansion of the promoter pool which could include 100s of promoters of different strengths. Likewise, the pool of available sgRNAs could also be broadened. Single nucleotide changes in the seed region of the sgRNAs could alter their affinities for their respective promoters, therefore altering repression efficiency and protein expression levels¹ .

On the other hand, the choice of proteins could be enriched from the vast repertoire of fluorescent proteins available. Additionally, fluorescent proteins could be substituted by any other protein and measured using other methods, for example by mass spec.

Our assembly method is entirely modular and inter-changeable, so any protein can be linked to any promoter and placed in any position in the plasmid. In this way, Key.coli can be easily optimised to serve a wide range of applications. Discernible combinations would only be limited by the reproducibility of signals and accuracy of the measurement.

Improving the design of the key coli transport device

The current design allows for revival at room temperature, however, since E. coli grows best at 37°C, future improvements will include a heating module surrounding the collection container at the base. The mechanism of this module is under discussion, but we favour the use of super cooled sodium acetate crystallisation, reducing the need for electricity in our key. This exothermic reaction will heat up the solution to the optimum temperature of approximately 30-35oC.

Also, to improve reproducibility and accurate authentication by the keyholder, the transport device could also incorporate a ‘reference’ strain, whose expression profile could always be used to normalise the fluorescence profiles obtained.

¹Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152(5), 1173–1183. http://doi.org/10.1016/j.cell.2013.02.022