Cell-based system
The cell-based design was developed by first visualising what types of functions a yeast cell would need to give a visual readout from a DNA sample. From the initial brainstorming session we realised that a yeast cell would need the following: A DNA detecting function, an inducible transcription factor, a gene which can produce colour without any substrate and an exponential system to make the readout more clear. To tackle these demands we found zinc-fingers as our DNA detecting molecule, these were coupled to a transmembrane protein. Two of these zinc-fingers were used together with an established membrane yeast two hybrid system using split Ubiquitin attached to the transcription factor LexA-VP16. This would make it so that if a sample of the correct DNA was found by a yeast cell, it would induce the transcription factor LexA-VP16 which would turn on two genes: MF(alpha)1 (a mating factor in yeast) and a Violacein gene (leading to the production of violacein, a coloured pigment which only requires aminoacids to be produced). The mating factor would make it so other yeast cells would also start expressing Violacein and the Violacein would turn the cells visibly violet.
The actual build of the cell-based system started of brilliantly, as the golden gate assembled parts starting forming, spirits were high and the preliminary results showed we had no mutations or misgivings in our construct. However, due to time limitations all the transformations weren't finished in time. The strain for testing the MF(alpha)1 system was finished in time though. The testing was done by measuring GFP expression using a plate reader and the results showed that the fluorescence was higher in these cells, indicating that it worked.
Cell-free system
The cell-free system was designed around a split TEV technique and splitting of the LacZ gene into an alpha fragment and an omega fragment. For the Alpha fragment, we used a previous iGEM teams Part: BBa_I732006 and the Omega fragment was designed in silico from Beta-galactosidase, deleting the specified amino acids according to their instructions [1]. We then fused the fragments with a cleavable linker in opposite order, which produces our Inhibited Beta-galactosidase protein (iGal). The split TEV technique we used was selected once we saw the results of the split luciferase in combination with a paired dcas9 that iGEM Peking 2015 used for tuberculosis detection. The Split TEV technique was taken from an article published in nature and there they had each half attached to two other proteins that could be induced to adhere to each other. [2] Combining these three resulted in our design: a system with a paired dCas9 which would create a functional TEVp to cut iGal to form a functional tetramer which would colour the sample blue in case our target DNA was present. See figure 2.
Building the project with golden gate assemblies was an arduous journey with many pitfalls among them are, PCR protocols that didn’t show results, gel extractions which went wrong, sequencing samples that got lost, and plate reading which were less than ideal. Eventually a final construct was made through the golden gate assembly, first all of the level one plasmids were transformed and grown in E. coli after which the level two assembly proceeded and multiplied in E. coli again. The parts were then genome integrated using Cas9 and the final construct was assembled in yeast.
Testing the parts of the cell-free system gave strange results, we can see that most samples are blue but not all and the different iterations of iGal and TEV seem to interact in ways that are unpredictable. The next step forward is to perform the experiment with purified proteins as there were unknown factors that made the negative controls give a positive response, which made it not possible to evaluate if the cell-free system works.
References
[1] http://parts.igem.org/Part:BBa_I732006
[2] https://www.nature.com/articles/s41589-018-0181-6