Team:Linkoping Sweden/Design
Background
Although approximately 70% of the world's surface is covered in water, as little as 0.025% of it is freshwater that is accessible to us [1]. Simultaneously, the world population is exceeding 7.9 billion and it is consistently growing. This results in a constant expansion of integrated agriculture and today, farming alone accounts for almost 70% of the global water withdrawals, placing immense pressure on the freshwater resources we have at our disposal [2]. Subsequently, the world is facing a crisis caused by the freshwater shortage and the effects of it are already apparent in all parts of the world. It is severely affecting the agricultural prospects in developing countries. Even here in Sweden, the agricultural industry is experiencing hardships caused by the lack of freshwater [3].
There are various existing methods for desalinating seawater and brackish water that have been developed as a solution to the global problem. The most commonly used methods are multi-effect distillation (MED), multistage flash (MSF), vapour compression (VC) and reverse osmosis (RO). However, many of them consume tremendous amounts of non-renewable energy causing them to leave large environmental footprints. Furthermore, the methods that do use renewable energy sources are tremendously more expensive than the ones using non-renewable energy. This means that neither economic nor environmental sustainability is achievable with the available desalination methods. Our project CyaSalt however, offers an environmentally friendly and economically accessible desalination solution to the freshwater shortage [4].
Design
To offer a method to desalinate seawater, we aim to use modified halophilic and phototrophic organisms to create freshwater for agricultural use. Our solution follows the CyaSalt cycle, illustrated in Figure 1. The process starts with the cultivation of the modified phototrophic organisms and then continues to the desalination of the saline water by the organisms. Thereafter, it proceeds to the separation of the phototrophic organisms from the desalinated water. The CyaSalt cycle ends with part of the leftover biomass being used for inoculation of the new cultures, and the remainder could possibly be used for biofuel production [5].
Cultivation
The desalination process is initiated with the cultivation of modified organisms. It is vital that these organisms survive in seawater for them to successfully execute the desalination. In order for the desalination process to be driven by sunlight and not require any external energy, they should also be photosynthetic. Based on these criteria, we worked with the halophilic and photosynthetic cyanobacteria Synechocystis sp. PCC 6803. These well-studied photosynthetic organisms have a high tolerance to salt and they have a naturally desalinating effect when growing in seawater [6,7]. To experimentally verify their growth in saline water, Synechocystis sp. PCC 6803 was grown in coastal water from Gotland and Gothenburg containing 1% and 3% salt with access to open atmosphere and light, as can be seen in Figure 2. They were simultaneously grown under the same conditions, but in BG-11 instead, to enable comparison. BG-11 is the standard growth media used when cultivating Synechocystis sp. PCC 6803 [8].
Desalination
A system consisting of four parts that are to be introduced to a phototrophic organism was designed to ultimately achieve desalination
To create an influx of chloride ions, we constructed a part containing the gene for halorhodopsin, an inward-directed chloride pump originating from the archaea Natronomonas pharaonis. halorhodopsin is activated by light and transports chloride ions into the cells against the concentration gradient as Figure 3 illustrates [9,10]. It has been used in Synechocystis sp. PCC 6803 by a previous iGEM team from Shanghai in 2015 to desalinate water. Their results showed a decrease in both the sodium and chloride ion concentration in the surrounding water which encouraged our choice to use halorhodopsin [11].
The second part integrated into the phototrophic organisms is the gene coding for channelrhodopsin from Chlamydomonas reinhardtii. Channelrhodopsin is, like halorhodopsin, activated by light. When activated, it transports sodium ions into the cells following the negative membrane potential created by the influx of chloride ions, as seen in Figure 4 [10].
The actions of halorhodopsin and channelrhodopsin are alike in multiple ways. In addition to them both being activated by light, they both require All-trans retinal to be bound to them to function. Thus, the access to All-trans retinal is a limiting factor for the chloride and sodium ion influx created by halorhodopsin and channelrhodopsin. All-trans retinal is formed when beta-carotene is cleaved by beta-carotene 15,15'-dioxygenase as Figure 5 illustrates. For this reason, our system includes a part that generates an excess expression of beta-carotene 15,15'-dioxygenase from Synechocystis sp. PCC 6803 in addition to halorhodopsin. This way, more beta-carotene can be cleaved so that the chloride ion influx is not limited by the lack of All-trans-retinal [12].
To avoid cell lysis due to the osmotic pressure created by water following the ion influx, the last part included in the desalination system is a Large Conductance Mechanosensitive Ion Channel, abbreviated MscL, from Synechocystis sp. PCC 6803. This is an ion channel that regulates osmotic pressure in the cells, and its purpose in our desalination system is to hinder cell disruption, as shown in Figure 6 [13].
Separation
After sodium and chloride ions have been imported into the cells, we wanted to create an easy way of separating the cells from the desalinated water. To achieve that, we designed a part that enables the cells to bind to a cellulose filter and thereby be separated from the water. This part consists of a fusion protein with two genes separated by a linker and is illustrated in Figure 7.
The first part is a membrane-bound S-layer protein called slr1272. Due to it being endogenous to Synechocystis sp. PCC 6803, slr1272 has a signal peptide sequence that guides it to the correct position in the membrane of that bacteria [14]. However, the fusion protein is designed so that slr1272 can be exchanged for another membrane-bound protein that is specific to another organism, making its applications more versatile.
The other part of the fusion protein is a carbohydrate-binding domain, also known as CBD. CBD has a very high affinity towards cellulose and will therefore bind strongly to a filter containing cellulose when desalinated water, containing bacterial cells, is passed through it [15-17]. This design creates a way of separating the cells containing the imported sodium and chloride ions from the desalinated water in an energy minimizing fashion.
In our separation construct, slr1272 and CBD are separated by a short glycine rich (-ASGGGGGG-). This linker also includes a NheI site (coding for -AS-) to enable cloning. The purpose of this is to position CBD further away from the other parts in the fusion protein so that it can be correctly folded without close proximity to the cell membrane or other proteins. The high glycine content in the linker is due to glycine not being very reactive, causing it to not be a target for many proteases. This creates a flexible random coiled linker [18].
Expression validation
To facilitate easy expression confirmation of our parts, we designed a part containing a fluorescent protein. This part contains almost the same linker sequence (-GSGGGGGG-) as the separation construct attached to a Green Fluorescent Protein (GFP). It was designed so that it can be attached to all the other parts we designed as Figure 8 demonstrates (by using the BamHI site which codes for -GS- in the linker). This way, the part that GFP is attached to will exhibit fluorescence [19]. This enables us to, by measuring GFP fluorescence, see which colonies have expressed our part. Furthermore, we can look at it under a microscope and thereby determine where in the cell our part is positioned.
[1] National Geographic. Freshwater crisis [Internet]. United States: National Geographic; [cited 2021 October 16]. Available from: https://www.nationalgeographic.com/environment/article/freshwater-crisis
[2] The World Bank. Water in Agriculture [Internet]. United States: The World Bank; [updated 2020 May 08; cited 2021 October 17]. Available from: https://www.worldbank.org/en/topic/water-in-agriculture#1
[3] Jordbruksverket. Jordbruket och vattnet [Internet]. Sverige: Jordbruksverket; [updated 2021 February 10; cited 2021 October 17]. Available from: https://jordbruksverket.se/jordbruket-miljon-och-klimatet/jordbruket-och-vattnet#h-Saarbetarvimedjordbruketsvattenhantering
[4] Karagiannis IC, Soldatos PG. Water desalination cost literature: review and assessment. Desalination. 2008 Mar;223(1-3):448-56. doi: 10.1016/j.desal.2007.02.071
[5] Sarsekeyeva F, Zayadan BK, Usserbaeva A, Bedbenov VS, Sinetova MA, Los DA. Cyanofuels: biofuels from cyanobacteria. Reality and perspectives. Photosynth Res. 2015 Aug;125(1-2):329-40. doi: 10.1007/s11120-015-0103-3. Epub 2015 Feb 22. PMID: 25702086.
[6] Pade N, Hagemann M. Salt acclimation of cyanobacteria and their application in biotechnology. Life (Basel). 2014 Dec 29;5(1):25-49. doi: 10.3390/life5010025. PMID: 25551682; PMCID: PMC4390839.
[7] Wurl O, Bird K, Cunliffe M, Landing WM, Miller U, Mustaffa NIH, Ribas-Ribas M, Witte C, Zappa CJ. Warming and Inhibition of Salinization at the Ocean's Surface by Cyanobacteria. Geophys Res Lett. 2018 May 16;45(9):4230-4237. doi: 10.1029/2018GL077946. Epub 2018 May 12. PMID: 29937608; PMCID: PMC6001423.
[8] van Alphen P, Abedini Najafabadi H, Branco Dos Santos F, Hellingwerf KJ. Increasing the Photoautotrophic Growth Rate of Synechocystis sp. PCC 6803 by Identifying the Limitations of Its Cultivation. Biotechnol J. 2018 Aug;13(8):e1700764. doi: 10.1002/biot.201700764. Epub 2018 May 2. PMID: 29577667.
[9] Amezaga JM, Amtmann A, Biggs CA, Bond T, Gandy CJ, Honsbein A, Karunakaran E, Lawton L, Madsen MA, Minas K, Templeton MR. Biodesalination: a case study for applications of photosynthetic bacteria in water treatment. Plant Physiol. 2014 Apr;164(4):1661-76. doi: 10.1104/pp.113.233973. Epub 2014 Mar 7. PMID: 24610748; PMCID: PMC3982732.
[10] Zhang F, Vierock J, Yizhar O, Fenno LE, Tsunoda S, Kianianmomeni A, Prigge M, Berndt A, Cushman J, Polle J, Magnuson J, Hegemann P, Deisseroth K. The microbial opsin family of optogenetic tools. Cell. 2011 Dec 23;147(7):1446-57. doi: 10.1016/j.cell.2011.12.004. PMID: 22196724; PMCID: PMC4166436.
[11] Team:SJTU-BioX-Shanghai/Design - 2015.igem.org [Internet]. 2015.igem.org. 2021 [cited 13 October 2021]. Available from: https://2015.igem.org/Team:SJTU-BioX-Shanghai/Design
[12] Culligan EP, Sleator RD, Marchesi JR, Hill C. Metagenomic identification of a novel salt tolerance gene from the human gut microbiome which encodes a membrane protein with homology to a brp/blh-family β-carotene 15,15'-monooxygenase. PLoS One. 2014 Jul 24;9(7):e103318. doi: 10.1371/journal.pone.0103318. PMID: 25058308; PMCID: PMC4110020.
[13] Sukharev, S., Blount, P., Martinac, B. et al. A large-conductance mechanosensitive channel in E. coli encoded by mscL alone. Nature. 1994 Mar;368:265–68. doi: 10.1038/368265a0
[14] Huang F, Hedman E, Funk C, Kieselbach T, Schröder WP, Norling B. Isolation of outer membrane of Synechocystis sp. PCC 6803 and its proteomic characterization. Mol Cell Proteomics. 2004 Jun;3(6):586-95. doi: 10.1074/mcp.M300137-MCP200. Epub 2004 Feb 26. PMID: 14990684.
[15] Tormo J, Lamed R, Chirino AJ, Morag E, Bayer EA, Shoham Y, Steitz TA. Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose. EMBO J. 1996 Nov 1;15(21):5739-51. PMID: 8918451; PMCID: PMC452321.
[16] Kruus K, Lua AC, Demain AL, Wu JH. The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9254-8. doi: 10.1073/pnas.92.20.9254. PMID: 7568112; PMCID: PMC40963.
[17] Dumitrache A, Wolfaardt G, Allen G, Liss SN, Lynd LR. Form and function of Clostridium thermocellum biofilms. Appl Environ Microbiol. 2013 Jan;79(1):231-9. doi: 10.1128/AEM.02563-12. Epub 2012 Oct 19. PMID: 23087042; PMCID: PMC3536079.
[18] Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct;65(10):1357-69. doi: 10.1016/j.addr.2012.09.039. Epub 2012 Sep 29. PMID: 23026637; PMCID: PMC3726540.
[19] Zimmer M. Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev. 2002 Mar;102(3):759-81. doi: 10.1021/cr010142r. PMID: 11890756.