What is bioHYDRA?

BioHYDRA is a project to create biological artificial muscles that respond to changes in humidity. This past year, Chen et al. [1] 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 wanted to improve 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. By affixing the ends of these HYDRAs to a given substrate, we could produce a folding mechanism that not responds to humidity, but is also reversible.

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

In our search for a folding mechanism for biOrigami, we wanted to find a something that wasn't a material, but a biological agent that could contract and at the same time have high resistance to harsh environments. That is how we first met bacterial spores. But, bacterial spores are usually thought of as inert, hibernating organisms with little to no metabolic function. So how can these actually be useful to us?
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. Suddenly, we have a folding mechanism. Thus, we sought to improve on the work of Chen et al. (Columbia University) and their HYDRA technology (Fig. 2), which is able to scale up the nano scale contractions of spores to the macro scale. We wanted to create fully biological HYDRAs, using cellulose and cellulose binding domains on the spore coat.

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Fig. 1 Scanning Electron Microscope picture of spores from our lab

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Fig. 2 HYDRAs in parallel (Chen et al.) [1]

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.

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Fig. 3 Spores on one of our HYDRA samples

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 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
Our results show promising initial results for future teams to expand on this project.

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Fig. 4 HYDRA in a humid and dry environment over the span of ~1 min. The HYDRA is only functional for the leftmost 3 cm, hence why measurements were only taken over that length

Vid. 1 HYDRA from 50% humidity to ~0% humidity (in the desiccator)

Recreating HYDRA: We were able to successfully create HYDRA by expanding on the work of Chen et al. . This was done as a proof of concept that this technology works and can be easily reproduced in a small lab setting. We constructed full lengths HYDRAs as described in Chen et al. [1].
Fig. 4. above shows our first functional HYDRA. On the left is a video of the HYDRA in action.

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.
We then transformed our construct into B. Subtilis. This was our main setback for this project, as many protocols did not seem to function. We believe that our 8 kb construct was very large and thus was hard to transform. After trying Xylose competence induced cells and electroporation, we decided to use the LMU Munich's MNGE transformation protocol. by picking certain colonies and undergoing colony PCR, we found that certain colonies seem to contain our part, which was confirmed by sequencing. Below is some data showing differential absorption between our control (aeBlue-) and our two samples which contain the insert (aeBlue+), using a spectrophotometer.
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Pic 5. Absorption spectra of our transformed spores (aeBlue+) and wild type spores (aeBlue-).

While absorbance at 597 nm (characteristic to aeBlue) does not show, there is a higher overall absorbance despite having the same spore concentration. Further testing is being done to determine the cause of this absorbance.

We also undertook a cellulose binding assay using scanning electron microscopy. We used four samples:
1. Wild type spores without a PBS wash
2. Spores with our contstruct without a PBS wash
3. Wild type spores with a PBS wash
4. Spores with our contstruct with a PBS wash
All spore samples were diluted to the same concentration of 10^9 spores/ml
Here are our results:
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Fig. 6 a. Wild type spores without a PBS wash (35 cells). b. Wild type spores with a PBS wash (10 cells). c. Spores with our construct without a PBS wash (463 cells). d. Spores with our construct with a PBS wash(93 cells). e. Graphical representation of cells remaining on each sample.

We can see that there is not only a net difference between washing and not washing the cellulose. But even more importantly, we can see that our construct yields a greater amount of spores adhering to the cellulose. These pictures were taken as representative of each sample, and thus these preliminary cell counts are accurate enough to show a statistical difference between cells that contain our construct and cells that do not. A PBS wash should get rid of all cells on the cellulose, but we can see that our CIPA+ spores are able to remain on the cellulose despite this wash, whereas the wild type CIPA- spores are almost fully washed out. This shows that our construct works.

Future Goals : From our work and the work done by Chen et al. , we know that synthetic HYDRAs function and can produce a large amount of force (10MJ/cm^3) [1]. Next steps would include building a biological HYDRA. While we have started to characterize our part, further tests would need to be done. We would need to evaluate why the spores do not show a distinct blue color from its aeBlue domain. Then, to better quantify cellulose binding, we would need to assay using absorbance left after x number of washes. After this characterization is finished, we would then reconstruct a HYDRA with cellulose and spores. If we succeed, the bioHYDRAs could then be used as a reversible, biological, self-folding mechanism for biOrigami. In conjunction with bioplastics, we can then create structures that contain both reversible and irreversible folds, expanding the potential and complexity of the structures we make with biOrigami.


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. Generic placeholder image

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

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.

Cloning: All our cloning was done with standard BioBrick cutsites. Our part was synthesized by IDT.

Spectrophotometry of spores for aeBlue Absorbance:

1. Determine spore concentration using microscopy and a hemocytometer.
2. Dilute spore suspensions to the same concentration (we used 1*10^9 cells/ml).
3. Add 200 µl of each sample into the wells of a 96 well plate.
4. Run a whole spectrum analysis.

CipA-cellulose binding assay using microscopy:

A - Cellulose Binding

1. Dilute spore suspensions to an equal concentration.
2. Spin down the spore suspensions and re-suspend in PBS.
3. Add 1.5 ml of spores PBS suspension to 2 ml tubes.
4. Cut a small piece of bacterial cellulose (BC) and add it to the 2 ml tubes containing the spores.
5. Let the 2 ml tubes sit for 1h at room temperature.

B - Washing and imaging

1. Fill 2 ml tubes with 1.5 ml PBS.
2. Take our the cellulose from the spore - PBS suspension and place in the new PBS 2 ml tubes
3. Slightly shake for 20 minutes.
4. Extract the cellulose and place in fume hood to dry.
5. Image through scanning electron microscopy.

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[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.

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