Team:BNDS China/Human Practices


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Human practice Aiming to find a way to increase the efficiency of the enzymes including RhlA, RhlB and RhlC, we design a way of mutating genes in order to increase production rate of rhamnolipid. To get our optimal enzyme, it involves multiple steps for us to achieve it including gene mutation and bacterial selection. In order to achieve each step, we have consulted many organizations and experts in the related industry. Through the talk with Dr. Li Lijun, an expert for improving enzymes degrading rhamnolipid in Jimei University. We’ve compared the ways to improve enzymes and asked her about the validity of our experiment. We’ve also got to understand the difficulties that we may face during our experiment. These connections largely helped us in conducting improvements for our design and making reflections of our project. Overview Q&A 1. Is it possible for you to give us some advice about increasing the activity of the enzyme through your experience? How do we choose enzymes for hydrolyzing rhamnolipid? A: It is really difficult to find an enzyme for hydrolyzing rhamnolipid. The structure of rhamnolipid may be rigid meaning that it may not be hydrolyzed. It also depends on where did you find this rhamnolipid. As a common example, cell walls can be made out of pectin which at the very end it will attach a rhamnolipid. I think you should research more about the enzymes on journals. If you do succeed, I will be glad you helped me to find out a new application for my enzyme. 2. May we ask how to improve the affinity between the chassis and the enzyme? A: Different enzymes have different characteristics. It is hard to give you a general explanation about how to improve your enzyme. But it will be better if you can use protein engineering to modify this enzyme. 3. We found that Dr. Li has used ways including point mutation, molecular stimulation and directed evolution for enhancing the activity of enzymes. Could you please share with us about your experience of modification of enzymes? A: As you did in your experiment, directed evolution is beneficial that you do not focus on a single point. Instead, you will get a bunch of results. In case of my early experiments, a total number of ten to the 36th power of enzymes have been created. It is better for you to use antibiotics to filter out what you want. Moreover, it will be better if you add a part of designing enzymes. Overall, it is beneficial for you to use a pre-designed enzyme and directed evolution. This would probably give you the best result. Suggestion Dr. Li also comments on our project design, she suggests that to increase the activity of the three enzymes, the rate-limiting enzyme activity must be increased first. Also, the rational design of the enzymes encouraged to be combined with the EvolvR system. To start with Point mutations, and then use it as the starting point to do directed evolution. Integrated Human Practice For integrated human practice, we visited and interviewed two professionals in the field of Biological Oil Recovery: Dr. Hu and Dr. Lin from Microbial Oil Recovery Research Center of Shandong Shengli Oilfield, Sinopec, and gain precious suggestions for technical problem and future direction from them. Current Progress on Biological Oil Recovery Biological Oil Recovery(BOR) refers to using the biological product, commonly biosurfactants, to recover oil. Compared to chemical reagents, Biosurfactants are more resistant to high temperature and salinity; this overall stability enables biological agents to withstand the extreme conditions of oil fields. Besides, the production of chemical agents requires energy that comes from the oil, so it’s less efficient and unfriendly to the environment. More importantly, some chemicals involved in the process are harmful to people. Thus there’s a couple of reasons for using BOR. By using it, the cost of fermentation of biological agents will be way lower, and sometimes food waste, like fermented molasses, can also be used. These organic substances will eventually be degraded by the environment when buried into the ground. The whole process should be pollution-free, green and low-cost. In all, using BOR will bring us lots of technical advantages: wide range of adaptation, simple process, low cost, good economic benefits, zero pollution. the Emulsifying abilityRhamnolipid is capable of emulsion recovery: the cysts formed by the hydrophilic head out and hydrophobic tail from biosurfactants- a structure similar to the phospholipid bilayer, can encase oil in its cavity and carry it out. Figure.1 during the interview Industrial Use: Character of Interest In practices, two properties used to evaluate the efficacy of a biosurfactant. Two important properties are used to evaluate the efficacy of biosurfactants. Concerns about Safety issuesIn synthesis process, since the bacteria that are being used now are all conditional bacteria like Pseudomonas aeruginosa, which mainly infects wounds, the danger is considered small. In addition, the post-processing of crude oil eliminates the potential danger of leakage. Wettabilitythe ability or tendency to spread over a solid surface. Rhamnolipid can change the wettability of underground rocks and gravel, on whose surface oil is stored. This change can largely reduce the interfacial tension, and thus improve the crude oil recovery rate. The professor also has pointed out a problem and gave us some advice for the future. In the production of Rhamnolipid at an industrial scale, the factors to be considered include the composition and proportion of the fermentation formula, the culture temperature during the fermentation process, the optimized temperature, the optimized PH value, the optimized inoculation amount of the whole seed fluid, the optimized oxygen, carbon, nitrogen and phosphorus and the optimized concentration of inducer. In fermentation, Rhamnolipid can produce a lot of foam. The foam has to drain out. Otherwise, it will affect the space. But a lot of the surfactants are actually in this foam. So the whole yield may be compromised if absorb the foam out. Therefore, how to prevent it from foaming or how to collect the foam has become the problem.For the advice for the future, the professor suggested some ways to increase production. First, we need to increase the synthetic precursor or promotor and remove the stimulating metabolites to boost output. Also, we need to transfer the gene for hemoglobin to the microbe, which will improve the entire organism’s ability to absorb oxygen. Figure.3 Fermentation techiniques employed by Sinopec Our Improvement Afterward After the visit to the research center, we made some improvements to our project. We adopted the suggestion for quantitatively testing Rhamnolipid by using HPLC-MS. In addition, thanks to Dr. Lin and Dr. Hu sharing one strain of P. aeruginosa that has been used in industry to produce Rhamnolipid with our team. Genetic modification is made on this strain of P.A., in order to increase its production of Rhamnolipid. Please see more details of our design and results in the following section. Figure.2 Illustration of the Emulsifying sbility
Remark: All activities in Human Practice are conducted in accordance with the regulation of iGEM,
and all consent forms are collected properly.

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