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| − | <h1 id="main-title">Human Practices</h1>
| + | <h1 id="main-title">Improve</h1> <br><br> |
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| − | <div id="Silver">
| + | <p><b><a href="http://parts.igem.org/Part:BBa_K2627000">http://parts.igem.org/Part:BBa_K2627000</a></b> <br> |
| − | <h4>Silver</h4><br>
| + | This year we made an improvement on part <a href="http://parts.igem.org/Part:BBa_K592001">BBa_K592001</a> , which codes for green/red light sensing protein CcaS. CcaS functions in CcaS-CcaR two-component system responding to the existence of green or red light. CcaS consists of the following domains and regions: a N-terminal transmembrane helix, a sensor domain consisting of a cyanobacteriochrome-type cyclic guanosine monophosphate phosphodiesterase/adenylyl cyclase/formate hydrogen lyase transcriptional activator (GAF) domain, a linker region, two period/aryl hydrocarbon receptor nuclear translocator/single-minded (PAS) domains of unknown function, a second linker region, and a C-terminal HK domain (Fig. 1) <a href="#r1"><sup>[1]</sup></a>. Previously, the functions of some of the domains were studied, for example, a shape change can be observed in the GFA domain after CcaS was exposed to light, which could thus induce an autophosphorylation of the HK domain, then the phosphate group would be transferred to the regulator protein CcaR<a href="#r2"><sup>[2]</sup></a>. However, the function of PSA domains is remained unknown. |
| − | <p><strong>Feasibility of our project</strong> <br>
| + | <br></p> |
| − | (1) This year we are interested in the application of optogenetic tools, and we desire to use them as our switch to redirect the metabolic flux. But whether optogenetic tools could be used as an ideal switch should be questioned. To verify that optogenetic tools can function as a probable switch, we consulted our primary PI Professor Liang, who majors in switches and their application in synthetic biology, before we started the experiment. | + | <img src="https://static.igem.org/mediawiki/2018/8/85/T--SDU-China--improve.png" alt=""> <br> |
| − | Professor Liang listed several switches that commonly used in synthetic biology such as quorum sensing, their functions, characters as well as usage. He emphasized that an element tool in synthetic biology must be measured several characters before being applied as an ideal switch. Firstly, a switch must have the ability to both active and repress the expression of the target gene, and the change between activation and repression should be fast enough and reversible. Besides, a switch should be stable at a relatively wide range of the concentration of inducers, and it should be easy to operate the switch. | + | <p class="reference"><b>Figure 1.</b> A brief illustration of the structure of CcaS. TF domain is located in the N-terminal while HK domain shows where the C-terminal is.</p> |
| − | Professor Liang also helped us evaluate whether optogenetic tools are capable of being a switch to regulate a genetic circuit. He told us optogenetic tools were highly suitable for a switch for their precise spatiotemporal resolution and precise dynamic control.
| + | <br> |
| | + | <p> We were inspired by Professor Koji’s work[1] in which he removed the two PSA domains to see what would happen. In his work, he created 11 variants of CcaS by deleting different numbers of base pairs, and these variants were characterized through measuring fluorescence/OD. We repeated his work and acquired this new part. This improved part is #4 in the variants collection. We found that this new part has a totally opposite response to green and red light compared with the original CcaS. As we can see from the figure below, #4 has a high fluorescence/Abs600 under red light but has a low one under green light.</p> |
| | + | <img src="https://static.igem.org/mediawiki/2018/2/26/T--SDU-China--improve2.png" alt=""> <br> |
| | + | <p class="reference"><b>Figure 2. </b>Characterization of part BBa_K2627000 and BBa_K592001. Part BBa_K2627000 shows a different effect after induced by green light and red light.</p> |
| | + | <br><br> |
| | + | <h4>References</h4> |
| | + | <p class="reference"> |
| | + | [1]. Nakajima, M., et al., Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal. Scientific Reports, 2016. 6(1). |
| | <br> | | <br> |
| − | <img src="https://static.igem.org/mediawiki/2018/5/52/T--SDU-China--metabolight.jpg width="100%" " alt="">
| + | [2]. Hirose, Y., Shimada, T., Narikawa, R., Katayama, M. & Ikeuchi M. Cyanobacteriochrome CcaS is the green light receptor that |
| − | (2) After certificating that optogenetics is capable for being an ideal switch, we began to take the compatibility of optogenetics and metabolic engineering into consideration.
| + | induces the expression of phycobilisome linker protein. Proc. Natl. Acad. Sci. USA 105, 9528–9533 (2008). |
| − | To explore the further utilization of optogenetics in metabolic engineering, we invited Yang Yi, the professor of East China University of Science and Technology to illuminate us the potential advantages of light-induced system compared with chemical-induced system. According to Professor Yang, such chemical-induced systems as Lac operon are not compatible with non-model host such as cyanobacteria, which are considered as competent candidates to produce biofuel and biomaterial through photosynthesize. Conventionally reported bacterial gene expression systems are based on the induction by specific chemicals, such as isopropyl β-D-1-thiogalactopyranoside (IPTG) and metal ions, which are not practical considering the large-scale cultivation of the cyanobacterial process. In addition, chemical inducers are difficult to be removed from the culture medium, making them unsuitable for downstream water recycling process. Therefore, an alternative gene expression system specific for cyanobacterial bioprocess should be developed. Thus light-controlled system applied to metabolism engineering provides a new a strategy to induce the behaviour of non-model host. <br></p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/7/7b/T--SDU-China--yangyi.jpg" width="500px" alt="">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/e/e7/T--SDU-China--yangyi2.jpg" width="500px" alt=""> <br>
| + | |
| − | <p> Professor Yang also introduce us the light-systems applied to mammalian cell, highlight the current application in the field of neuroscience. <br>
| + | |
| − | When it comes to the hardware of the light-controlled system, which we worried about a lot, Professor Yang provided us with the resource of the light device applied in his project. And he also suggested that since the light-controlled system is highly sensitive, the hardware is not that demanding. However, due to its high sensitivity, we must pay much attention to the impact of natural light which is inevitable while operating experiments.
| + | |
| − | <br></p>
| + | |
| − | | + | |
| − | <p> (3) What’s more, we participated in CCiC 2018 held in Shanghai, China and had the opportunity to exchange ideas with many other teams and we derived a lot of suggestions from others to increase the feasibility of our project. <br></p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/9/96/T--SDU-China--ccic1.jpg" height="400px" alt="">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/e/ef/T--SDU-China--ccic2.jpg" height="400px" alt=""> <br>
| + | |
| − | <p>
| + | |
| − | We met a member from Peking and he had a project related to VVD (an optogenetic tool which can sense blue light). He mentioned that the element sensing blue light was extremely sensitive to light, even if just little natural light existed could influence the stability of it and even could come with a reverse effect, so we would better operate our blue light sensing system in darkness or under red light. <br>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/e/eb/T--SDU-China--ccic3.jpg" width="800px" alt=""> <br>
| + | |
| − | We also met a member from XMU-China, who came up with a valuable suggestion to our project. According to his words, the culture medium used in industrial fermentation and that in lab fermentation were totally different. In factories, cost needs to be taken into consideration, so the nutrients in industrial culture medium come from the waste produced by other industries. Thus, industrial culture medium could be non-transparent which doesn’t allow light to go through and reach the bacteria.
| + | |
| − | </p> <br>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/5/57/T--SDU-China--ccic4.jpg" height="400px" alt="">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/a/a1/T--SDU-China--ccic5.jpg" height="400px" alt="">
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| − | </div>
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| − | <div id="integrated">
| + | |
| − | <h4>Gold&Integrated</h4>
| + | |
| − | <p><strong>Introduction</strong> <br>
| + | |
| − | | + | |
| − | According to the expertise we’ve got, we know that optogenetics, used as a switch, has a bright future in metabolic engineering, which means our idea is valuable and practicable. Let’s turn our sight onto this new system itself.
| + | |
| − | Since our goal is to introduce a new and convenient inducer—light into fermentation industry, the core of our project is that how to put the new system into practice. Since the system is promising in its marketing value with both the new inducer and environmental-friendly product, we cannot be satisfied with completing building this system and testing it in our lab, what we need to do is applying this system into practical use, our system finally should be tested in the real factory. However, there are great differences between fermentation in labs and industrial fermentation, before putting this new system into practice, we should figure out the distinctions between fermentation in labs and factories, in this way we can find some guidance in improving our system to break these obstacles. <br>
| + | |
| − | | + | |
| − | <strong>What we did </strong><br>
| + | |
| − | To figure out the differences, we had an unforgettable tour this summer to an industrial fermentation factory to visit and learn something about fermentation. We visited a biotechnology company called Weifang KDN Biotech, in Weifang, Shandong Province. We mainly visited several workshops there. <br>
| + | |
| − | Different workshops with different usage were located in the industrial park, the largest one, equipped with huge, spheroid fermenter, was the main place where fermentation took place. Raw materials, as well as engineered bacteria, were stored in the fermenters. While fermentation, special device would check and regulate temperature and pH inside the fermenters to ensure the environment was suitable for bacterial growth and production. Once fermentation was ended, the bacteria liquid would flow to another workshop through many pipes. Next, the product should be separated from bacteria and other liquid, the theory base of product purification is kind of like chromatography. Finally, the waste would be poured into treatment tank and experienced special treatment. <br>
| + | |
| | </p> | | </p> |
| − | <img src="https://static.igem.org/mediawiki/2018/2/2a/T--SDU-China--gongchang1.jpg" width="400px" height="300px" alt=""> | + | < !--<img src="https://static.igem.org/mediawiki/2018/e/ e2/T--SDU-China-- chara.png" alt="">--> |
| − | <img src="https://static.igem.org/mediawiki/2018/d/d7/T--SDU-China--gongchang2.jpg" width="400px" height="300px" alt="">
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| − | <p> <br>
| + | |
| − | We also consulted their skilled workers about the process of fermentation and the usage of equipment, and we posed our idea and light-induced control of metabolic flux system. Our questions were answered in detail and the workers also gave some advice on our light- induced system. <br><img src="https://static.igem.org/mediawiki/2018/e/ ed/T--SDU-China-- gongchang3.jpg" width="800px" alt=""> | + | |
| − | <br><br>
| + | |
| − | <strong>What we found</strong><br>
| + | |
| − | According to the human practice we did and the theoretical knowledge we collected, we found that there are still some defaults existing in our project, we must figure out the problems and break those obstacles. Now, we are going to list the obstacles we need to overcome.<br>
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| − | <a href="#aspect1"><img src="https://static.igem.org/mediawiki/2018/0/02/T--SDU-China--btn.png" width="130px" alt=""></a>
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| − | <div><b>Culture Medium</b></div>
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| − | <div><b>engineered bacteria</b></div>
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| − | <div><b>Hardware</b></div>
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| − | <div><b>Cost</b></div>
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| − | <p ><br><br>
| + | |
| − | <b class="aspect" id="aspect1">1. culture medium. </b><br>
| + | |
| − | As we mentioned before, culture medium applied in industrial fermentation and lab fermentation is of great difference. In labs, we commonly use nutrient-rich culture medium such as LB or other culture medium with glucose as carbon source. This kind of culture medium looks transparent that allows light to go through the liquid and reach the bacteria. So that we can easily operate the light-induced system. However, in a factory, engineered bacteria should be cultured in seed medium before getting into the fermentation medium. Seed medium is used to expand the scale of the seed bacteria so the nutrient inside is rich to accelerate the growth of bacteria. Seed medium sounds like the culture medium we use in our lab. Fermentation medium, although, contains the basic nutrient carbon source; nitrogen source; inorganic salts; water and growth factors, it also needs precursors and promoters. Precursors and promoters are required to promote the production of products. Besides, we need to concern about the efficiency that the bacteria take carbon and nitrogen source into usage. When bacteria take nutrient at a relative low speed, bacteria growth will slow down and energy will be stored for the next fermentation. Moreover, the composition in fermentation medium is not precise in chemicals, the raw materials of fermentation often come from wastes from other industries to low down the cost, and usually the raw materials are commonly insoluble, which means there will be solid particles inside the medium, causing shading inside fermenters. In this case what we need is to optimize fermentation medium. To achieve optimization of culture medium, the ratio between fast-usage carbon/nitrogen source and slow-usage carbon/nitrogen source should be proper considering the balance between growth and production, C/N should also be suitable for fermentation with different types of engineered bacteria, and the source of raw material much be accessible with a low cost. <br>
| + | |
| − | <b class="aspect" id="aspect2">2. industrialized engineered bacteria</b><br>
| + | |
| − | A type of engineered bacteria born in lab should meet some requirements so it can live in a fermenter and do its duty. <br>
| + | |
| − | <b>First of all</b>, high efficiency of production is required for engineered bacteria. Not only the engineered bacteria should be efficient in producing products, but the efficiency of purification is also important. Purification of intracellular products is much more difficult than that of extracellular products, for the reason that cell lysis is the prerequisite of purification and there are lots of other proteins, nucleic acids, polysaccharides, and even if lipids. So engineered bacteria producing extracellular product will increase the profits dramatically.<br>
| + | |
| − | Secondly, the compatibility between culture medium and chassis should be tested before industrial fermentation. Normally, chassis used for fermentation including <i>E. coli</i>, Bacillus, yeast and fungi must be compatible with the culture medium, especially the carbon source. Glucose, which is a monosaccharide, is now widely used as carbon source in fermentation, glucose is easily absorbed by chassis but can be poisonous to chassis when the concentration is high, so chassis that can tolerant high concentration of glucose will be more suitable for fermentation.<br>
| + | |
| − | <b>Besides</b>, safety of chassis and operation is of great importance in selecting engineered bacteria. Pathogens and conditional pathogens are not allowed for fermentation unless the pathogen genes are knocked out. And the chassis for fermentation is forbidden to leave the fermenters, to prevent contamination to the environment.<br>
| + | |
| − | <b>What’s more</b>, engineered bacteria should be easy to operate to face the change in environment. During fermentation, engineered bacteria will produce a large quantity of byproducts, which may influence or even repress bacteria growth and synthesis. Therefore, we should analyse the relativity among bacteria growth, bioreactor operation and environment to regulate metabolic flux inside the cells and optimize fermentation.<br>
| + | |
| − | <b>Last but not least</b>, stability in heredity is also of great importance to engineered bacteria. Exogenous genes responsible for production is better inserted onto bacteria chromosomes than onto a plasmid. As to genes must be located on a plasmid, stability should come to the first place before constructing the engineered bacteria, resistance to only one antibiotic is better than that to multiple antibiotics.<br>
| + | |
| − | <b class="aspect" id="aspect3">3. Hardware</b><br>
| + | |
| − | According to the trouble caused by non-transparent culture medium, we must make some developments in the light devices. First of all, we need to solve the problem that light cannot reach our bacteria, so that we can think of further improvement even if application. However, putting light into practical use may need a great innovation in fermenter as well as other equipment, the light devices should be equipped onto the fermentation and be connected to the regulating equipment. As we all know an innovation in the structure of fermentation as well as other equipment could cost a lot of time and money, and this innovation suffers a risk of low profit but high cost (we will discuss it later). <br>
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| − | <img src="https://static.igem.org/mediawiki/2018/e/e8/T--SDU-China--gongchang4.jpg" height="300px" alt=""> <br>
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| − | <b class="aspect" id="aspect4">4. cost</b><br>
| + | |
| − | The cost of the whole process for a type of new engineered bacteria as well as a new inducer transferring from labs to factories should be analysed and we should make a prediction to imitate the comparison between costs and profits. Raw materials for culture medium, maintenance of fermenters and regulating equipment, introduction of new light devices and purification of products, all these parts require money and should be in our consideration.<br>
| + | |
| − | </p>
| + | |
| − | <p><strong>Conclusion</strong> <br>
| + | |
| − | In this summer, we have built a new awareness about metabolic engineering and fermentation, which reminds us of the drawbacks of our project design and experiment. Overall, if we desire to introduce our project to industrial production, we have to solve all the problems listed above. Recently, we have optimized the carbon source in our fermentation medium by replacing glucose with glycerol, and we also have made a model to analyse how different carbon source, nitrogen source and inorganic salt in culture medium can affect the production of PHB as well as other metabolic pathways. But that is not enough, what we need to do in the future is to accomplish the whole project and optimize the chassis, light devices, culture medium and flexibility of the whole project.<br></p>
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