Team:UNIK Copenhagen/Design
COSMOCROPS
The co-culture - device and design
Introduction
A co-culture refers to a consortium of two or multiple species of microorganisms living together, interacting in a “social-network” sometimes helping, at other times inhibiting, each other. They act a lot like roommates actually. When living together, you communicate, you interact, and you regularly affect the mood of your mate, for better and for worse.
You cooperate with your roommate too, which is also the goal of our co-culture system; to make Synechococcus elongatus produce the needed carbon source for a culture of Bacillus subtilis to grow, facilitating the production of a target compound by B. subtilis through cooperation.
The target compound could potentially be anything in the biobrick registry, however, we have chosen to focus on usable compounds for space exploration and settlement. For this project specifically, we sought to facilitate the production of the bioplastic, poly-lactic-co-3-hydroxybutyrate acid (PLA), using novel biobricks within B. subtilis.
Figure 1: Our provider strain. In nature, S. elongatus is often found in well-lit marine environments. For CosmoCrops 2016 the strain PCC7942 of S. elongatus was used. Reprinted with the permission of “Biopix”(http://www.biopix.dk/) © JC Schou.
Figure 2: Our manufacturer. B. subtillis is a gram positive bacteria found in soil and in the microbiota of animals. The strain used in CosmoCrops 2016 is B. subtilis 168.
Building on top of existing knowledge
As an interdisciplinary team of many skills, we wanted to work with astrobiology and bioprocessing because it played to our interests, and because we could make a difference to the future of mankind. We also wanted to expand on a former project in iGEM, to further develop on a great idea, and to put principles into practice that had not been done before. We would therefore like to thank the iGEM team of Brown-Stanford 2011 for recognizing the value of cscB permeases in cyanobacteria (ref. 1), and the work of Daniel Ducat at Pamela Silver’s Lab (Brown-Stanford 2011). We also wish to thank the iGEM teams of Amsterdam 2014 and Nevada 2011 for the inspiration of using co-cultures and dialysis bags respectively.
Why co-cultures suit space exploration better than monocultures
Bioproduction generally offers a much needed method of production in space that could lower the requirements to spacecraft cargo, both in terms of diversity and volume, allowing for longer and more elaborate space mission. Basically, the more you can produce in space from extraterrestrial resources, the less you have to bring with you. For instance, bioproduction and synthetic biology could be used to produce fuel, medicine, plastic, food and nutritional supplements from very scarce or simple resources like sunlight and carbondioxide(CO2). However, bioproduction comes with a cost, modern production facilities utilizing monocultures often require large and costly equipments which are simply impractical to bring along with you in space. Furthermore, one would have to bring several bioreactors to allow for continuous production.
By utilizing two organisms instead of one, we envision that astronauts can use a single bioreactor to produce a plethora of useful end products, either in parallel or one after the other. The ultimate goal being to reduce the amount of tools a space pioneer would need to bring along with him or her to facilitate biological based production, and, at the same time, enabling space pioneers to produce any goods needed, when needed. We envision, a cartridge-based system where astronauts use a closed system of containers with the described compartments inside (fig. 3), the compartments are made switchable to allow for easy extraction and change of manufacturer-strain.
Figure 3: Closed system of two exchangeable compartments for co-culturing in space. Green and yellow compartments contain S. elongatus and B. subtilis respectively. Extra inlets were included to make room for additional supplementation like carbondioxide. The compartments are exchangeable because a dialysis membrane separates the two compartments.
Providing a feedstock for B subtilis - Making of the provider strain
In principle, our co-culture design (fig. 4) relies on there being a “provider”, and a “manufacturer” that thrives in the feedstock/environment supplemented by byproducts of the provider’s metabolic activity. We utilized S. elongatus (PCC7942) of the cyanobacterial phylum as a provider, because of its photosynthetic ability and tendency to accumulate sucrose in its intracellular environment in response to osmotic stress (Hagemann et al. 2011).
However, the bacteria lacks a way to export the sucrose to the surrounding media. To fully utilize the accumulated sucrose, we relied on previous research in rerouting metabolic carbon fluxes to the extracellular space. This work was done by Daniel Ducat at Harvard Medical school in the lab of Pamela Silver where PCC7942 was transformed with the cscB sucrose/proton symporter from E.coli (Ducat et al. 2012). To accelerate our mission we utilized the original cscB-transformed PCC7942 strain with permission from the original authors.
To validate the sucrose-exporting capabilities of transformed S. elongatus and characterize a previously made biobrick inspired from Dr. Ducat’s research (BBa_K656011), the cscB-transformed PCC7942 was grown and induced to export sucrose by 150 mM NaCl and 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) with and without chloramphenicol (CAM) as measured by High-Throughput-Liquid-Chromatography (HPLC). Sucrose accumulated to maximum concentrations of 1.4 mM after a week of stimulation. To guarantee that 150 mM was the optimum concentration of NaCl to support sucrose export other NaCl concentrations were tested aswell, fromm 0-500 mM (experiment 1).
|
Validation / Characterization of BBa_K656011 |
Results |
Link |
|
1) Does the cscB-transformed PCC7942 export sucrose? How much? |
Positive |
Figure 4: Co-culture principle. An inner compartment made from a repurposed dialysis bag (2 kDa, Spectra/Por ® 6 dialysis membrane, 132633) contains B. subtilis producing a product. The outer compartment contain S. elongatus secreting generating a carbon source for B. subtilis to live on. The dialysis bag serves both as mechanical separation and easy extraction of the product without cross contamination. Edited and reprinted with permission of Gert Gram and Jyllands-Posten. Along with the model, a flask with LB with 10 ug/ml CAM as been incubated overnight with inoculated mock-transformed B. subtilis isolated in the inner compartment.
Enabling production from a common feedstock
The choice of B. subtilis as “manufacturer” strain, was made taking into account its general resistance to hazardous environments (Moissl-Eichinger et al 2016), and its ability to form hazard-resistant endospores (Errington J. et al 1993 and Bucker et al. 1974). Endospores, at least theoretically, could be utilized to create a library of transformed hibernating bacteria that upon addition of growth media can resurrect through germination, growing, and producing a wanted compound. We believe that such a system would make transportation less expensive, avoiding the need of cryogenic storage, and maybe more importantly, enabling the possibility of a closed system where astronauts could avoid contact with the bacterial cultures directly because all initial protocols are prepared on earth. Such closed system would be highly relevant to avoid contamination of the recycled atmosphere in extraterrestrial environments such as space stations, where opportunistic bacteria have been observed to thrive at the risk of the astronauts’ wellbeing (Chechinska 2015).
Productive cooperation
Cooperation between roommates can sometimes be challenging, and for natural enemies such as two different bacterial species, it can seem almost impossible. Microorganisms are often involved in a battle of dominance fighting for space and nutrients in the confined space where they live. To reduce such competition, we repurposed a dialysis-bag to create two compartments mechanically separating the subcultures (fig. 3). Mind that the final goal would be to design cartridges to separate provider and manufacturer strains by dialysis membranes, but due to time constraints, design validation was based on a simpler model using a growth flask and an immediately available dialysis-membrane from Spectrum Labs (2 kDa, Spectra/Por ® 6 dialysis membrane, 132633)(fig. 4). We utilized Dextran Blue, a polymer-based dye with a molecular weight of approximately 2 million Da, to characterize the integrity of our membranes. The membranes did not leak the dextran blue solution after five days, and the dialysis-bag was therefore regarded as leak-proof (experiment 2). Furthermore, we tested our inoculation protocol in rich-medium(LB-medium) to characterize and optimize our sterilization steps. Our sterilization steps were successful, enabling us to isolate the inner compartment (inoculated with B. subtilis) from the outer compartment (not inoculated) (experiment 3).
|
Validation |
Results |
|
|
2) Was it possible to create a semi-permeable compartment by using a dialysis membrane? |
Positive |
|
|
3) Was it possible to keep a subculture growing inside the isolated compartments without contaminating the surroundings? |
Positive |
Finding the right media composition
--- See protocol tap for all media recipes ---
The first phase of the project was to determine a suitable media composition for B. subtilis and S. elongatus.
S. elongatus was found to readily grow on BG-11 (experiment 4), a common minimal media for S. elongatus and used in Ducat. et al. 2012 (Ducat et al. 2012). B. subtilis was also tested for growth on BG-11, however no growth was observed across all groups (experiment 5), which confirmed that our co-culture could not be based on BG-11 alone. This lead us to try other medium compositions for B. subtilis (experiment 6), from which ATCC was further characterized. During these experiments we observed that B. subtilis grows in sucrose-supplemented ATCC medium, but require additional supplementation in certain time intervals to sustain positive growth (experiment 7), in our experiments, we supplemented every fourth day with new medium. Furthermore, B. subtilis also undergo sporulation in absence of sucrose and growth rates drop tremendously (experiment 7). In conclusion, ATCC was chosen because it allows for B. subtilis growth on supplemented minimal media, and sporulation in absence of sucrose.
|
|
|
|
|
4) Can S. elongatus grow in BG-11 media? (S. elongatus minimal media). |
Positve |
|
|
5) Can B. subtilis grow in BG-11 media with and without sucrose supplements? |
Negative |
|
|
6) Testing B. subtilis in ATCC, CSE and C'-minimal media |
Inconclusive |
|
|
7) Can B. subtilis grow on ATCC with and without supplemented sucrose? |
Positive |
A detail worth noticing is the fact, that Ducat. et al. 2012 (Ducat et al. 2012) used 150 mM NaCl to induce sucrose accumulation in S. elongatus. To investigate whether such osmotic stress would be harmful to B. subtilis growth assays with B. subtilis in 50, 100, 150, 200, and 500 mM supplemented NaCl LB-medium was made. From this, we observed a minor growth inhibition at 500 mM NaCl, but without major consequences at 200 mM and below (experiment 8). A similar growth experiment with S. elongatus at different concentrations of NaCl was also undertaken showing a lower growth rate at 200 mM NaCl (experiment 9).
|
|
|
|
|
8) Can B. subtilis grow under osmotic stress? |
Positive |
|
|
9) Can S. elongatus grow under osmotic stress? |
Positive |
To make sure that both bacteria could grow our in co-culture under experimental conditions, we hypothesize that a mixture of ATCC and BG-11 in a 1:1 ratio could enable cultivation of both. To test this, B. subtilis and S. elongatus was investigated in mixed medium under experimental conditions. B. subtilis was readily able to grow in ATCC:BG-11 media supplemented with 30 mM sucrose as expected (experiment 10). Both WT and cscB-transformed S. elongatus grew in BG-11:ATCC mixed medium, however, the transformed strain clearly grew slower than WT (experiment 11). Furthermore, when IPTG was added the cscB-transformed strain would drop in OD, suggesting activation of cscB leads to exit of log-phase (experiment 8). This also suggests, that sucrose export is growth limiting, which was also observed in Ducat et al 2012 (Ducat et al. 2012). The reason for this could be due to cscB rerouting carbon flux to the extracellular matrix inhibiting carbon metabolism and growth. For future research, it would be important to design a strain of S. elongatus that can sustainably export a carbon source and sustain its own population simultaneously.
|
|
|
|
|
10) Can B. subtilis grow in ATCC:BG-11 mixed medium with supplemented sucrose? |
Positive |
|
|
11) Can S. elongatus grow in ATCC:BG-11 mixed medium? Can S. elongatus grow during induction (+150 mM and 0.1 IPTG) as it should in our final setup? |
Negative, a future challenge |
Design validation - Using spent media to simulate the co-culture environment
Finally, to make a convincing device validation we had to not only characterize our media, but also test whether it could work as a co-culture. We knew from previously described sucrose measurements, that cscB-transformed S. elongatus were indeed able to increase the concentration of sucrose in the media, the spent BG-11 media, the media of activated cscB-transformed S. elongatus, would thus have a carbon source for B. subtilis to use, hence, we expect it to grow more. To validate, whether B. subtilis could grow in the final environment of our co-culture, spent medium (BG-11 + sucrose) was mixed with ATCC at a 1:1 ratio, 10 ug/ul chloramphenicol (CAM) was supplemented, and CAM-resistant B. subtilis was inoculated into the suspension culture. The growth measured in OD(600) was compared between spent medium from WT and cscB-transformed S. elongatus(experiment 12). The results were positive, showing a clear increase in growth between uninduced transformed B. subtilis, IPTG induced transformed B. subtilis and WT. This implicates that our co-culture can be better than a monoculture under the same conditions, however, our project still lack an ability to measure an output/product, without which a direct estimation of cost-benefit between monocultures and co-cultures in space, is near impossible.
|
|
|
|
|
12) Can B. subtilis grow in spent BG-11 media from cscB transformed S. elongatus? |
Positive |
Device validation - Using our co-culture to produce plastic
Due to issues with the genetic construct, which is further commented in the tap on “biosynthetic make-up”, we could not assemble our biobrick construct in due time. This also prevented us from using our co-culture to produce plastic. However, if we had had the time to do so, the following experiment would have been undertaken (experiment 13).
|
Experiment |
|
|
|
13) Is our composite biobrick able to produce plastic? |
Not started |
Conclusion
We propose to use co-cultures as a mean of bio-processing in space, because of its potential to reduce the required cargo-load compared to monoculturing. Furthermore, it enables the design of a sustainable and modular system of bioprocessing consisting of a producer (S. elongatus) and a manufacturer (B. subtilis) that can be extended to produce multiple end-products in parallel with each other. We tested the ability of these specific bacteria, due to their photosynthetic capabilities and endospore formation of S. elongatus and B. subtilis respectively. B. subtilis could be grown in supplemented ATCC:BG-11 mixed medium, but at lower growth rates than supplemented ATCC media, and was very resistant to osmotic stress. S. elongatus could also be grown in mixed medium, however, did not tolerate high osmotic stresses and cscB-sucrose export showing loss of growth rate in presence of IPTG and 150 mM NaCl. To validate our co-culture model, we used spent media of cscB-transformed and induced S. elongatus to simulate the environment of the co-culture. From validation, we conclude that B. subtilis grow with higher rates in spent media - which should allow for a more productive B. subtilis culture compared to a monoculture in the same media without additional supplements.
References
2011 Brown-Stanford working on "Powercell". https://2011.igem.org/Team:Brown-Stanford/PowerCell/Introduction
Martin Hagemann. Molecular biology of cyanobacterial salt acclimation, FEMS Microbiology Reviews. 2011;35(1)87-123.
Ducat DC, Avelar-Rivas JA, Way JC, Silver PA. Rerouting Carbon Flux To Enhance Photosynthetic Productivity. Applied and Environmental Microbiology. 2012;78(8):2660-2668.
Christine Moissl-Eichinger, Charles Cockell, Petra Rettberg. Venturing into new realms? Microorganisms in space, FEMS Microbiology Reviews Sep 2016, 40 (5) 722-737
Errington J. Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiological Reviews. 1993;57(1):1-33.
Bucker, H. et al. Viability of Bacillus subtilis spores exposed to space environment in the M-191 experiment system aboard Apollo 16. Life Sci Space Res 1974;12:209-13.
Checinska A, Probst AJ, Vaishampayan P, et al. Microbiomes of the dust particles collected from the International Space Station and Spacecraft Assembly Facilities. Microbiome. 2015;3:50.
