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{{SJTU-BioX-Shanghai/Header | title = Desalination Process}}
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==Process design ==
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===Three-stage cultivation===
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{{ SJTU-BioX-Shanghai/Header | title = Design}}
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==Design of transport module==
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The cultivation of engineered cyanobacteria is proposed to be comprised of three stages: growth stage, induction stage (starvation stage) and desalination stage. And here are the reasons of this design:
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Halorhodopsin (HR) proteins are light-driven inward-directed chloride pumps from halobacteria. They are membrane-integral proteins of the rhodopsin superfamily that form a covalent bond with the carotenoid-derived chromophore all-trans retinal. Absorption of a photon with a defined optimal wavelength induces trans-cis isomerization of retinal, which triggers a catalytic photocycle of conformational changes in the protein, resulting in the net import of one chloride per photon into the cytoplasm. (Amezaga,J.M.et al.2014) We use it as our biodesalination driver which confers cyanobacteria the ability to absorb chloride to a significant degree.
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1. Since high density of cyanobacteria is essential for efficient biodesalination and expression of heterologous protein will probably decrease the growth rate significantly, there should be a growth stage between the inoculation time and the beginning of induction.
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As for sodium, cyanobacteria can actively export sodium ion to maintain low internal sodium concentration under saline environment. And this is mainly performed by Na+/H+ antiporter and P-type Na+ ATPase; the former one requires H+ gradient as the energy source and the latter one consumes ATP directly. Since the ATP synthase is driven by H+ gradient, we can simply regard that ATP is required for active Na+ export. And the ATP requirement provides opportunity to halt sodium export by depleting internal ATP stores. Manipulation of cultivation conditions such as omitting photosynthetically efficient wavelengths from the light spectrum will may deplete nutrient supply and exhaust ATP reserves. Moreover, we propose that the negative membrane potential generated by halorhodopsin would drive the influx of cation through sodium ion channels.
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2. In our biodesalination process, the induction stage is also starvation stage because the induction conditions we designed omitted main energy source of cyanobacteria-red light.
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{{ SJTU-BioX-Shanghai/Figure/f1.2.1 }}
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3. Halorhodopsin is a light-driven chloride pump, and the maximum absorption wavelength is 580nm, which locates in the yellow light region. Therefore the light condition for halorhodopsin to work must be different from the condition for the induction stage.
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In summary, the halorhodpsin drives Cl- inside, thus generating negative membrane potential, which will drive import of Na+ given that starvation inhibits active Na+ export. This design of transport module is shown in Figure 1.2.1 .
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===Process controlled by PcpcG2===
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==Design of control system ——PcpcG2 and Pdark==
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Based on PcpcG2 and halorhodopsin, we established a biodesalination system which relies on red and green light. The light colors in the three stages are red light, green light and white light respectively.
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Light-inducible and heavy metal inducible promoters are the main biological switches in synthetic biology of cyanobacteria. Considering that our ultimate goal is to obtain an engineered organism capable of extracting sodium chloride from seawater without leaving any harmful substance, candidate promoters should meet a few key criteria. Firstly, the inducing condition shouldn’t leave any harmful substance that is difficult to remove. Secondly, the inducing condition shouldn’t cost too much if applied to industrial scale. Thirdly, the inducing condition should be compatible with starvation. Based on these conditions, we selected two light-inducible promoters which are described in the following paragraphs.
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In the growth stage, red light is enough for photosynthesis of cyanobacteria, which has been proved to be feasible by a recent report(Abe, K. et al. 2014). To further confirm it, we determined the growth curve under red light, which is shown in Figure 1.4.1. This growth rate is no slower than that of the stain under natural light as long as we adjust the light intensity.
| label = Growth curve of Wild-Type ''Synnechosystis sp.''strain PCC 6803 under red light.
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| descr = 3ml of culture (OD = 0.5) was inoculated into 50ml BG11 medium at the 0 h. All error bars indicate standard deviations(n=3).
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The control of PcpcG2 over downstream gene is based on the CcaS-CcaR-PcpcG2 system identified in PCC 6803, which has been characterized and further engineered to be a useful genetic tool. (Abe, K. et al. 2014) The phycobilisome linker gene cpcG2 is chromatically regulated by the sensor histidine kinase CcaS and cognate response regulater CcaR. CcaS catalyzes antophosphorylation followed by phototransfer to CcaR under green light[4]. Phosphorylated CcaR binds to PcpcG2 and induces expression of cpcG2, or halorhodopsin in our recombinant strain. The control mechanism is seen in Figure 2.
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In the induction stage, the green light induces the expression of HR. Additionally, green light can’t provide energy source for chlorophyll, thus creating a starvation condition. And the starvation can inhibit active export of sodium, which is essential for biodesalination in the working stage.
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In the working stage, halorhodopsin absorbs light to drive chloride import. Addtionally, cyanobacteria will regain energy under natural light. The biodesalination process controlled by PcpcG2 is shown in Figure 1.4.2.
| label = Biodesalination process controlled by PcpcG2.
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| descr = In the growth stage, cyanobacteria absorb energy from red light and grow to a certain density; In the induction stage, green-light induces the expression of halorhodopsin, and additionally pushes cyano bacteria to starvation status; In the working stage, engineered cyanobacteria absorb sodium chloride under natural light and cyanobacteria regain energy.
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}}</center>
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Red light and green light require artificial illumination or extensive light filter, which may cause difficulty in scaling up biodesalination process. We searched for another promote that allows the process to rely only on natural light and darkness. And Pdark, which is from previous iGEM team (BBa_K1026009), was our answer. This “dark-sensing” promoter combines PcpcG2 and a constitutive promoter from Ecoli and it can be regarded as the “reverse PcpcG2”. Pdark contains binding site of CcaR in PcpcG2, which overlaps with the binding site of RNA polymerase. Therefore the binding of RNA polymerase to Pdark will be blocked and transcription can’t be initiated when phosphorylated CcaR binds to it. The control mechanism of Pdark is seen in Figure 3.
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===Process controlled by Pdark===
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Based on Pdark and HR, we constructed an improved biodesalination system which depends only on the switch between white light and darkness. In the growth stage and working stage, we provide white light, while in the induced expression stage the light source is removed. Darkness leads to starvation and the starvation can inhibit active export of sodium, which is essential for biodesalination in the working stage.(described l in section transport module). The biodesalination process controlled by Pdark is shown in Figure 1.4.3.
| label = Biodesalination process controlled by Pdark.
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| descr = In the growth Stage, cyanobacteria grow to a log-phase; in the induction stage, darkness induces the expression of halorhodopsin, and additionally pushes cyanobacteria to starvation status; in the working stage, engineered cyanobacteria absorb sodium chloride under natural light.
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}}</center>
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==Biodesalination Assay==
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===Assay on PcpcG2-HR===
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==Design of biodesalination process==
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The effectiveness of this biodesalinaion process is proved by determination of the concentrations of extracelluar sodium and chloride or desalination assay, which are shown in Figure 1.4.4. During the early time of working stage, there is an obvious decrease of concentration compared to that of Wild-type, which indicates that our biobrick (PcpcG2-HR, BBa_K1642010) really works in cyanobacteria under this biodesalination process! The following rise of concentration can be explained by the regain of energy under natural light in the working stage. Considering that we focus on the biodesalination process controlled by Pdark, we didn’t optimize this process.
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The cultivation of engineered cyanobacteria comprises three phases: growth phase, expression phase and desalination phase. After cyanobacteria reaching a high density, we induce the expression of halorhodopsin, and subsequently we move the cyanobacteria into white-light condition afterwards, which allows the halorhodopsin to work.
| label = Biodesalination assay of the process controlled by PcpcG2.
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| descr = The start of induction stage is at -12h and the times of taking samples are at -12h, 4h, 12h and 24h.
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}}</center>
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Based on PcpcG2 and HR, we established a biodesalination system which relies on red and green light. The light color in the three stages are red light, green light and white light respectively. In the growth stage, red light is enough for photosynthesis of cyanobacteria and we determined the curve under red light (seen in the Experiment and Results section). In the induced expression stage, the green light induces the expression of HR. Additionally, green light can’t provide energy source for chlorophyll, thus creating a starvation condition. In the working stage, halorhodopsin absorbs light to drive chloride import.
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===Assay on Pdark-HR===
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Based on Pdark and HR, we constructed an improved biodesalination system which depends only on the switch between white light and darkness. In the growth stage and working stage, we provide white light while in the induced expression stage we remove the light source. Moreover, if the induced expression stage is as long as the night, this improved biodesalination system can be control by the normal changes between day and night without any human intervention.
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The effectiveness of this biodesalination process is proved by determination of the concentrations of extracellular sodium and chloride or desalination assay, which are shown in Figure 1.4.5. During the early time of working stage, there is an obvious decrease of concentration compared to that of wild-type, which proves the function of our biobrick(Park-HR, BBa_K1642011). An obvious decrease during the early time and a following rise are consistent with that of the process controlled by PcpcG2.
| label = Biodesalination assay of the process controlled by Pdark.
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| descr = The start of induction stage is at -12h and the times of taking samples are at -12h, 0h, 1h, 2h, 4h, 12h, 24h and 36h.
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}}</center>
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(Figure)
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To figure out the limitation of desalination, we prolonged the length of the induction stage and adjusted the times of taking samples. The results are shown in Figure 1.4.6. The 10h in the working stage is approximately the minimum point.
| label = Biodesalination assay of the process controlled by Pdark with more frequent sampling times.
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| descr = The start of induction stage is at -16h and the times of taking samples are at -16h, 0h, 2h, 2h, 4h, 6h, 8h,10h,12h.
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}}</center>
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{{ SJTU-BioX-Shanghai/Footer }}
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The acquisition of the minimum point makes it possible to design a longer biodesalination process. We can extend this process by alternating induction stage (starvation stage) and working stage, make the cyanobacteria to experience starvation and regain of energy for more cycles, thus achieving more reduction of salinity. Moreover, if the length of the induction stage and working stage are approximately 12h, after growth stage this improved biodesalination system can be controlled by the natural alternation between day and night without any human intervention
The cultivation of engineered cyanobacteria is proposed to be comprised of three stages: growth stage, induction stage (starvation stage) and desalination stage. And here are the reasons of this design:
1. Since high density of cyanobacteria is essential for efficient biodesalination and expression of heterologous protein will probably decrease the growth rate significantly, there should be a growth stage between the inoculation time and the beginning of induction.
2. In our biodesalination process, the induction stage is also starvation stage because the induction conditions we designed omitted main energy source of cyanobacteria-red light.
3. Halorhodopsin is a light-driven chloride pump, and the maximum absorption wavelength is 580nm, which locates in the yellow light region. Therefore the light condition for halorhodopsin to work must be different from the condition for the induction stage.
Process controlled by PcpcG2
Based on PcpcG2 and halorhodopsin, we established a biodesalination system which relies on red and green light. The light colors in the three stages are red light, green light and white light respectively.
In the growth stage, red light is enough for photosynthesis of cyanobacteria, which has been proved to be feasible by a recent report(Abe, K. et al. 2014). To further confirm it, we determined the growth curve under red light, which is shown in Figure 1.4.1. This growth rate is no slower than that of the stain under natural light as long as we adjust the light intensity.
Figure 1.4.1 Growth curve of Wild-Type Synnechosystis sp.strain PCC 6803 under red light. 3ml of culture (OD = 0.5) was inoculated into 50ml BG11 medium at the 0 h. All error bars indicate standard deviations(n=3).
In the induction stage, the green light induces the expression of HR. Additionally, green light can’t provide energy source for chlorophyll, thus creating a starvation condition. And the starvation can inhibit active export of sodium, which is essential for biodesalination in the working stage.
In the working stage, halorhodopsin absorbs light to drive chloride import. Addtionally, cyanobacteria will regain energy under natural light. The biodesalination process controlled by PcpcG2 is shown in Figure 1.4.2.
Figure 1.4.2 Biodesalination process controlled by PcpcG2. In the growth stage, cyanobacteria absorb energy from red light and grow to a certain density; In the induction stage, green-light induces the expression of halorhodopsin, and additionally pushes cyano bacteria to starvation status; In the working stage, engineered cyanobacteria absorb sodium chloride under natural light and cyanobacteria regain energy.
Process controlled by Pdark
Based on Pdark and HR, we constructed an improved biodesalination system which depends only on the switch between white light and darkness. In the growth stage and working stage, we provide white light, while in the induced expression stage the light source is removed. Darkness leads to starvation and the starvation can inhibit active export of sodium, which is essential for biodesalination in the working stage.(described l in section transport module). The biodesalination process controlled by Pdark is shown in Figure 1.4.3.
Figure 1.4.3 Biodesalination process controlled by Pdark. In the growth Stage, cyanobacteria grow to a log-phase; in the induction stage, darkness induces the expression of halorhodopsin, and additionally pushes cyanobacteria to starvation status; in the working stage, engineered cyanobacteria absorb sodium chloride under natural light.
Biodesalination Assay
Assay on PcpcG2-HR
The effectiveness of this biodesalinaion process is proved by determination of the concentrations of extracelluar sodium and chloride or desalination assay, which are shown in Figure 1.4.4. During the early time of working stage, there is an obvious decrease of concentration compared to that of Wild-type, which indicates that our biobrick (PcpcG2-HR, BBa_K1642010) really works in cyanobacteria under this biodesalination process! The following rise of concentration can be explained by the regain of energy under natural light in the working stage. Considering that we focus on the biodesalination process controlled by Pdark, we didn’t optimize this process.
Figure 1.4.4 Biodesalination assay of the process controlled by PcpcG2. The start of induction stage is at -12h and the times of taking samples are at -12h, 4h, 12h and 24h.
Assay on Pdark-HR
The effectiveness of this biodesalination process is proved by determination of the concentrations of extracellular sodium and chloride or desalination assay, which are shown in Figure 1.4.5. During the early time of working stage, there is an obvious decrease of concentration compared to that of wild-type, which proves the function of our biobrick(Park-HR, BBa_K1642011). An obvious decrease during the early time and a following rise are consistent with that of the process controlled by PcpcG2.
Figure 1.4.5 Biodesalination assay of the process controlled by Pdark. The start of induction stage is at -12h and the times of taking samples are at -12h, 0h, 1h, 2h, 4h, 12h, 24h and 36h.
To figure out the limitation of desalination, we prolonged the length of the induction stage and adjusted the times of taking samples. The results are shown in Figure 1.4.6. The 10h in the working stage is approximately the minimum point.
Figure 1.4.6 Biodesalination assay of the process controlled by Pdark with more frequent sampling times. The start of induction stage is at -16h and the times of taking samples are at -16h, 0h, 2h, 2h, 4h, 6h, 8h,10h,12h.
The acquisition of the minimum point makes it possible to design a longer biodesalination process. We can extend this process by alternating induction stage (starvation stage) and working stage, make the cyanobacteria to experience starvation and regain of energy for more cycles, thus achieving more reduction of salinity. Moreover, if the length of the induction stage and working stage are approximately 12h, after growth stage this improved biodesalination system can be controlled by the natural alternation between day and night without any human intervention
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