Abstract Why spores?

BioHYDRA is a project to create biological artificial muscles that respond changes in humidity. This past year, Chen et al. at Columbia University devised a way to utilize the power of evaporation and the way Bacillus spores expand and contract depending on ambient humidity in order to create contractile structures coined as “HYDRA” (Hygroscopy driven artificial muscles). We improved on this technology by creating fully biological hydras, using cellulose instead of polyimide, and incorporating cellulose binding sites on the spore coats instead of using artificial artificial glue.

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Introduction How spores and the Stanford-Brown 2015 iGEM team first met

In our search for a biological agent that can contract and at the same time have high resistance to the environment, we came across bacterial spores. Bacterial spores are usually thought of as inert, hibernating organisms with little to no metabolic function. In response to stressful environmental conditions, vegetative Bacillus subtilis will sporulate to produce robust organisms called spores, which can survive in extreme conditions for many years. To be able to germinate and regain full vegetative function, they have to maintain a certain internal environment so as to preserve the integrity of its organelles while in spore form. To regulate the humidity content in the spore core, B. subtilis have adapted by changing the shape of its wrinkled spore cortex with various degrees of humidity. As the air becomes dryer, the spores shrinks, and vice versa. We sought to improve on the work of Chen et al. (Columbia University) and their HYDRA technology (Pic. 1) to create fully biological HYDRAs, using cellulose and cellulose binding domains on the spore coat.

Pic. 1 HYDRAs in parallel (Chen et al.) [1]

Experiment From synthetic to biological

There were two experiments, recreating HYDRAs from the Chen et al. publication, and creating bioHYDRAS, which are fully biological versions of HYDRAs.

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.

Data and Results

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. 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.

Protocols

Making HYDRAs (from Columbia's Chen at al.: [1]

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. It is crucial to ensure that there is no surface on the polyimide tape that is not covered with spores.

Sporulation: [2]

Difco Sporulation Medium (DSM) Per liter:

Bacto nutrient broth (Difco) 8 g
10% (w/v) KCl 10 ml
1.2% (w/v) MgSO4·7H2O 10 ml
1 M NaOH ~1.5 ml (pH to 7.6)
Adjust volume to 1 liter with ddH20. pH to 7.6. Autoclave and allow to cool to 50°C.

Just prior to use, add the following sterile solutions (and antibiotics if required):

1 M Ca(NO3)2 1 ml
0.01 M MnCl2 1 ml
1 mM FeSO4 1 ml
Procedure:

1. Inoculate colony into 25 ml DSM and grow at 37°C and 150 rpm until mid-log phase 0.45 < OD600 < 0.6 (usually 2 hours).
2. Dilute 1 to 10 into 250 ml of prewarmed (37°C) DSM in 2L flask. Incubate a further 48 hrs at 37°C and 150 rpm. Observe culture occasionally during growth, and continue to next step if >90% of culture are free spores.
3. Centrifuge the culture 10 min at 10,000xg and carefully discard the supernatant.
4. Wash the pellet with 200 ml of cold (4°C) sterile distilled water. Centrifuge for 10 min
at 10,000xg and again discard the supernatant.
5. Resuspend the pellet in 200 ml cold distilled water and leave at 4°C overnight.

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).

[2] W. Nicholson & P. Setlow, in Molecular Biological Methods for Bacillus, eds. C. Harwood & S. Cutting, New York: John Wiley, pp.391-450, 1990.