Team:XMU-China/Design
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There have been numerous examples of capacity degradation of nuclear power plants due to clogging in the cooling water system caused by marine organisms. The blockage is usually on the grounds of adhesion and outbreak of sea life, which would be the barriers of water flux. The main method to deal with the adhesion and the breakout is chemical disinfection, which is destructive to the waters nearby, using sodium hypochlorite to inhibit or kill the larvae or spores of marine organisms and hamper their attachment and growth on the filters, the flow channels after the filtering device and the pipes of the coolers. In addition, physical means like manual cleaning and replacing of grilles are adopted, which is also rough and expensive.
After brainstorming and HP investigation, we focused on the threat posed by Phaeocystis globosa and Mytilus edulis, and decided to build a new kind of engineering bacteria characterizing precaution and emergency treatment to against the congestion caused by the P. globosa and the M. edulis.
SALVAGE, which is short for Scientifically and Ably Leverage Vibrio natriegens to Alleviate the problem of P. globosa and M. edulis, is a brand new means applying synthetic biological ways to remove the most haunting mussels and algae impacting on the nuclear power plants.
How we implement SALVAGE?
Why we choose synthetic biological methods?
The common way of removal mentioned above is quite hazard to the environment. Hence, it is of importance to seek for solutions environmentally friendly and durable to figure out the blocking issue. Synthetic biology, a neotype interdiscipline, features specificity, operability and practicability to help us handle this issue. Genes from different sources could be utilized to reform engineering bacteria to produce copious target functional substances continuously to achieve the goal we set. Comparing with the traditional and innovative measures, the synthetic biological one is selected to cope with the blockage led by marine organisms.
Vibrio natriegens
We chose Vibrio natriegens as the chassis bacteria, which could grow rapidly in the hyperosmotic environment with a generation time of less than 10 minutes1. Furthermore, the methodology now for genome editing and heterologous expression of Vibrio natriegens is rather routinization. Notwithstanding, the vast nutrient consumption owing to its rapid growth also places a demand on the design of our hardware.
Fig. 1. Currently established molecular biology techniques in V. natriegens. These methods comprise DNA transformation methods, such as electroporation, heat-shock transformation and conjugation, as well as genome engineering techniques, such as recombineering, multiplexed genome editing via natural transformation (MuGENT) and the use of the CRISPR-Cas9 system.
Phaeocystis globosa
Phaeocystis globosa is one of the most widely distributed algae causing red tides. The distinctive feature of P. globosa is its complex history of heterotypic life, which is characterized by morphological transformations between single cells and colonies. Once the colonies are formed, the protective glycosaminoglycan-rich membrane of the colonies would greatly enhance the resistance of P. globosa, which makes the removal of colonies and algae more difficult.
Fig. 2. P. globosa: from single cell to colonies
We enact the tactic that the Histidine ammonia-lyase (HutH, derived from Pseudomonas putida) is released into the surrounding of P. globosa and converted the L-histidine in the mucus to trans-urocanate, which then elevates the ROS in P. globosa to damage the algal cells3. This method would prevent the formation of algal bloom by inhibiting the growth of single cells of P. globosa. In addition, we choose cellulose on the cellular surface of P. globosa as the target, and use cellulose binding protein (CBM, derived from Clostridium thermocellum) to combine with HutH. The binding of CBM and cellulose ensures HutH kill the algal cells more precisely.
Fig. 3. Cellulose on the surface of P. globosa is recognized by the fused protein CBM-HutH which convert the histidine around to trans-urocanate
In order to make the design more comprehensive, solving the issue of the colonies is also required. The strategy is that using SpyTag with SpyCatcher and the strong galactose-involved polysaccharide binding protein (LecA, derived from Pseudomonas aeruginosa strain PAO1) to capture the vesicles of the colonies of P. globosa and then to form the hydrogel aggregation sinking to the bottom of the water environment4,5.
Fig. 4. Galactose on the surface of P. globosa is recognized by LecA attached to SpyCatcher-aggregated Spytag
Mytilus edulis
M. edulis, which is the most viscous mussels, also performs a notorious role in clogging nuclear plants. Owing to its own specific traits, the M. edulis are capable to attach the grilles, pipes and even the tubes of condensers, causing blockage. It works by the following mechanisms6: Firstly, the byssus of the mussels can adhere to solid surfaces and produce mussel foot protein(Mfp), and the interaction between the byssus and the surface is quite strong for the large number of DOPA in the side chains of Mfp can form hydrogen bonding with metal surfaces. Secondly, the byssus of the mussels can also release fatty acids to provide a hydrophobic environment to protect and promote the Mfp of its function. Therefore, to solve the adhesion issue of M. edulis mainly led by Mfp, the engineered bacteria we devise should execute the following functions: inhibition the formation of mussels’ byssus, removal of fatty acids and elimination the effect of DOPA.
The functional enzymes we need were determined by searching the literatures. The TnaA-his (BBa_K3739044) can convert tryptophan to indole7, which inhibits the formation of byssus in mussels. RhlA and RhlB (derived from Pseudomonas aeruginosa) can produce a kind of biological detergent, rhamnolipid, to remove fatty acids8. And PPO (polyphenol oxidase, derived from Bacillus megaterium) can prevent mussel adhesion by oxidizing the DOPA in the Mfp into dopaquinone9.
Fig. 5. Diagram of mussel adhesion and treatments
Signal peptides and Surface display system
In this project, to deal with the congestion of coarse grilles of the nuclear power plants, we designed an extracellular secretion system and a surface display system for Vibrio natriegens to prevent the outbreak of P. globosa and the adhesion of M. edulis. To guarantee the release of the target proteins expressed by Vibrio natriegens, two signal peptides, Aly01 and LMT, were elected to help us translocate these target proteins to the extracellular circumstance to unlock the subsequent reactions. It should be noticed that the signal peptide LMT was identified by us, see more information about this in our Resultpage.
Fig. 6. The process of secretion
For the surface display system, the three following proteins were picked, LCI KR-2, OmpA and LamB. LCI KR-2 is a type of peptide mutant from Bacillus subtilis with strong capacity of adsorption of polypropylene. Therefore, the crude grilles would be coated with the polypropylene and the Vibrio natriegens carrying LCI KR-2 could adhere to the surface of grilles for long-term release of polyphenol oxidase and rhamnolipids to prevent attachment of mussels. There would be a question that how could LCI KR-2 be present at the outerspace of the cells? OmpA and LamB come to help. OmpA and LamB are anchoring proteins that anchor their cargo proteins which here is LCI KR-2 to the exterior plasma membrane, so that LCI KR-2 could be displayed on the outer surface of Vibrio natriegens. As a result, Vibrio natriegens are capable of adhering to the grilles coated with polypropylene.
Fig. 7. Surface display system and LCI KR-2 is displayed on the outer surface of Vibrio natriegens
Kill Switch
The cooling water system of nuclear power plants is directly connected with the ocean, which cautions us about the potential risk of leakage of the engineered bacteria. In consideration of the biosafety, an effective kill switch is arranged for the whole framework. Because the engineered bacteria will be working in a dark aqueous environment, and to avoid the use of chemical inducers, a light-induced kill switch based on optogenetics is being developed, which can be activated by the irradiation of blue-light with certain wavelengths. To fit this design, blue-light strips would be paved at the dark regions either directly connected to the ocean or existing risks of leakage like water inlet and outlet of the circulatory system. Blue-light would trigger the death of the engineered bacteria before they enter natural environment.
Fig. 8. Circuit design of suicide switch
EL222
An optogenetic gene expression system based on the bacterial transcription factor EL222 is used to regulate the production of hazardous proteins. EL222 is a light-oxygen-voltage (LOV) protein (light-activated DNA-binding protein) with a light-sensitive LOV domain and an HTH (Helix-Turn-Helix) DNA-binding domain found in bacteria10. The LOV domain binds to the HTH domain in the dark, covering the HTH-4 helix, which is essential for DNA binding. Illumination with blue-light initiates a photochemical reaction between the LOV structural domain and its chromophore, allowing EL222 to dimerize and bind the pBLind promoter, leading to the transcription of downstream genes.
This system has advantages such as a simple inducing condition, a quick response time, a highly linear reaction sensitive to blue light intensity, and a large dynamic range of protein expression11. We created a pBLind-EL222-pBLind-blrA blue light-induced system by putting EL222 sequences downstream of promotor pBLind. The expression levels of EL222 and BlrA are low in the dark, minimizing material and energy waste as well as harmful protein leakage. When exposed to blue light, EL222 stimulates the transcriptional activity of pBLind, resulting in a positive feedback loop that boosts the expression of EL222 and the deadly protein BlrA, eventually killing engineering bacteria.
Fig. 9. EL222 activates pBLind promoter under blue light induction
BlrA
BlrA is a blue light-induced GTP-binding receptor and a flavin-binding fluorescence protein from Bacillus subtilis. It contains an optogenetic toolbox based on LOV photosensitizers. Under normal conditions, these photosensitizers are not toxic to cells, but when exposure to blue light, they generate reactive oxygen species that kill cells. The blue light density has to be greater than 90 mW/cm2 and the treatment time should be greater than 10 seconds to achieve a relatively high mortality rate12.
We've combined the toxic protein gene blrA with the EL222 photoinduced system in the hopes of building bacteria that can only survive in their designated area, and the target bacteria will be activated to suicide without damaging the environment once they enter the scope of blue light strips.
Fig. 10. Effect process of blue light induced suicide switch
1. Weinstock, M. T.; Hesek, E. D.; Wilson, C. M.; Gibson, D. G., Vibrio natriegens as a fast-growing host for molecular biology. Nat Methods 2016, 13 (10), 849-51.
2. Hoff, J.; Daniel, B.; Stukenberg, D.; Thuronyi, B. W.; Waldminghaus, T.; Fritz, G., Vibrio natriegens: an ultrafast-growing marine bacterium as emerging synthetic biology chassis. Environ Microbiol 2020, 22 (10), 4394-4408.
3. Zhuang, L.; Zhao, L.; Yin, P., Combined algicidal effect of urocanic acid,N-acetylhistamine andl-histidine to harmful algaPhaeocystis globosa. RSC Advances 2018, 8 (23), 12760-12766.
4. Kuhaudomlarp, S.; Gillon, E.; Varrot, A.; Imberty, A., LecA (PA-IL): A Galactose-Binding Lectin from Pseudomonas aeruginosa. Methods Mol Biol 2020, 2132, 257-266.
5. Yang, X.; Wei, J.; Wang, Y.; Yang, C.; Zhao, S.; Li, C.; Dong, Y.; Bai, K.; Li, Y.; Teng, H.; Wang, D.; Lyu, N.; Li, J.; Chang, X.; Ning, X.; Ouyang, Q.; Zhang, Y.; Qian, L., A Genetically Encoded Protein Polymer for Uranyl Binding and Extraction Based on the SpyTag-SpyCatcher Chemistry. ACS Synth Biol 2018, 7 (10), 2331-2339.
6. Kim, E.; Dai, B.; Qiao, J. B.; Li, W.; Fortner, J. D.; Zhang, F., Microbially Synthesized Repeats of Mussel Foot Protein Display Enhanced Underwater Adhesion. ACS Appl Mater Interfaces 2018, 10 (49), 43003-43012.
7. Wang, X.; Huang, Y.; Sheng, Y.; Su, P.; Qiu, Y.; Ke, C.; Feng, D., Antifouling Activity towards Mussel by Small-Molecule Compounds from a Strain of Vibrio alginolyticus Bacterium Associated with Sea Anemone Haliplanella sp. J Microbiol Biotechnol 2017, 27 (3), 460-470.
8. Chong, H.; Li, Q., Microbial production of rhamnolipids: opportunities, challenges and strategies. Microbial Cell Factories 2017, 16 (1).
9. Sendovski, M.; Kanteev, M.; Ben-Yosef, V. S.; Adir, N.; Fishman, A., First structures of an active bacterial tyrosinase reveal copper plasticity. J Mol Biol 2011, 405 (1), 227-37.
10. Jayaraman, P.; Devarajan, K.; Chua, T. K.; Zhang, H.; Gunawan, E.; Poh, C. L., Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic Acids Res 2016, 44 (14), 6994-7005.
11. Motta-Mena, L. B.; Reade, A.; Mallory, M. J.; Glantz, S.; Weiner, O. D.; Lynch, K. W.; Gardner, K. H., An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat Chem Biol 2014, 10 (3), 196-202.
12. Endres, S.; Wingen, M.; Torra, J.; Ruiz-Gonzalez, R.; Polen, T.; Bosio, G.; Bitzenhofer, N. L.; Hilgers, F.; Gensch, T.; Nonell, S.; Jaeger, K. E.; Drepper, T., An optogenetic toolbox of LOV-based photosensitizers for light-driven killing of bacteria. Sci Rep 2018, 8 (1), 15021.