Recreating HYDRAs: By expanding on the protocol by Chen et al. (1) to create HYDRAs, we then used desiccant and wet paper towels in separate chambers to create humidity variance for these HYDRAs to expand and contract, and recorded our results using a ruler and a humidity sensor.

BioHYDRAs: The goal of BioHYDRA was to replace all the parts of HYDRAs by biologically produced substances. We sought out to replace polyamide tape by bacterially cellulose, and the glue by cellulose binding domains on the surface of the spore coat. Thus, the first step involved cloning a Bacillus construct in Escherichia coli of a fusion protein sequencing consisting of a spore coat protein, cotZ (building off work done on Sporobeads by the LMU Munich 2012 iGEM team), and a cellulose binding domain (CIPA). Additionally, we decided to add aeBlue, a chromogenic protein, between cotZ and CIPA to be able to see with the naked eye whether Bacillus is in a vegetative or a spore state. The plasmid would thereafter need to be transformed and expressed in Bacillus . Here is a link to our part: BBa_K1692028 . We then needed to produce bacterial cellulose. For more details, refer to our Cellulose page. Finally, our project would consist of testing for the binding affinity of the spores on the cellulose before we could construct our bioHYDRAs. To do so, we used the cellulose binding affinity protocol that the 2015 Edinburgh team sent us in light of our collaboration.

Recreating HYDRA: We were able to successfully create HYDRA by expanding on the work of Chen et al. . We used 5µl of 0.1% Poly-L-Lysine to coat the polyamide tape on each spot where we wanted to put spores. We then allowed it to dry for several hours. A spore-glue mixture was made using 1µl of Elmer’s glue with 1 ml of a suspension of spores (2.55e9 spores/ml). 10µl of this spore glue mixture was applied to the poly-L-Lysine coated surfaces and allowed to dry. Preliminary results show small contraction of squares of polyimide tape as shown in Pic (insert number here). We constructed full lengths HYDRAs as shown in the diagram below, taken from Chen et al. (1).

BioHYDRA: We were able to successfully create the fusion protein cotZ-aeBLUE-CIPA in E. coli . This construct was ligated into the pSBbs1C backbone ( BBa_K823023). We then transformed E. coli to grow our plasmid. We are excited to have been able to use our newly developed CRATER technique to better select for our plasmid when transforming, which accelerated our project substantially. By running the plasmid through a gel and by sequencing, we were able to confirm that we had the right size (8 kb) and sequence for the plasmid.

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Protocols

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References

(1) Chen, X. et al. Scaling up nanoscale water-driven energy conversion into evaporation-driven engines and generators. Nat. Commun. 6:7346 doi: 10.1038/ncomms8346 (2015).