Team:FAFU-CHINA - 2020.igem.org

Part

We found a high expression level of promoter FBA1 when we reviewed the paper, and a team has expressed plasmids containing this promoter in S. cerevisiae in the past years. (Part number: BBa_K2765044,link: http://parts.igem.org/Part:BBa_K2765044) We further searched the literature to determine its basic promote strength.

Seung-pyo Hong et al^[1] constructed an expression vector containing FBA1 in Yarrowia Lipolytica (pDMW212 & pDMW214), and measured its GUS Reportor activity and mRNA expression.

Figure 2. Histochemical and ﬂuorometric GUS reporter assays to measure promoter activity. Fluorometric determination of GUS activity. GUSspeciﬁc activity was expressed as nM 4-methylumbelliferone released/min/mg protein

Figure 2. Relative quantities of GUS mRNA. The relative transcript levels were calculated using the GUS mRNA level from strains with TEF1::GUS as the reference. Assays were done in quadruplicate.

Troubleshooting

We have summarized the lessons learned from building the team until now, and have written a document to help future Igemer to avoid these problems.

(1) Experiment: Starting from the design of primers for dry experiments, the screening of wild strains, and the modeling of proteins for wet experiments,
(2) Modeling: There are some common models, such as the logistic equation most commonly used in the lab. For teams that prefer industrial tracks, Monod and Luedeking-Piret equations are also available. In addition, for the team that needs to express the enzyme, The Miesian equation is also a more commonly used model.
(3) HP: HP is a unique aspect of the IGEM.The judged Handbook is different every year, so ou need to keep an eye on what's happening to HP every year In addition to practical application research and interviews, it is difficult but significant for human practice to lead educational development and popularize synthetic biology concepts.
(4) Team Building: The international Genetic Engineering Competition is a biology discipline competition with high difficulty and high gold content. Faced with limited economic support, limited experimental period and limited experimental research, especially under the impact of COVID-19 this year, the negative impact of these problems will become more severe.; For public school students, you need to deal with school resources in case you drop out due to lack of funds
(5) Team management: It is strongly not recommended that free-Rider participate in this competition and abandon the mentality of not contributing anything but waiting for the award. A truly united team is one that coordinates, develops together and moves toward the same goal. In this case, the team members should be highly enthusiastic about the international genetic engineering competition and have the ability to arrange and plan tasks and time coordination with certain intensity.

Open source blueprint

When we were designing the fermentation tank, we found that many models of the fermentation tank could be used universally, but the average student had no access to get these drawings and had to buy them in the store. Here we have uploaded some CAD drawings of the open source fermentor we found this year for future Igemer to refer to when designing hardware

download CAD file

Reference：

[1] Hong S P , Seip J , Walters-Pollak D , et al. Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1IN promoter[J]. Yeast, 2012, 29(2).

Excellence in Another Area G#7

While improving our own project’s safety, we pay more attention to popularizing the public's awareness of experimental safety.

i. We visit many labs to find out the potential danger of their daily operations :

1. EB (Ethidium bromide): EB is mainly used for DNA fluorescence labeling during electrophoresis. This acridine dye is a strong mutagen. However, many operators in laboratories often touch EB glue by hands directly. Some students with the lack of experiment experience add excessive EB in order to achieve better color effect.

2. Trizol: Trizol is a commonly used reagent for RNA extraction. There is a large amount of phenol in Trizol, which is corrosive. However, many students do not wear laboratory suits, protective glasses and masks when doing RNA extraction.

3. The use of Clean Bench and Biosafety Cabinet: In most laboratories, many students use the Clean Bench directly after using UV light for sterilization without blowing out ozone. The Biosafety Cabinet sucks the air inside, while the clean Bench blows the air outside. The misuse of the Clean Bench instead of the Biosafety Cabinet may cause harm to laboratory personnels, such as the use of immunohistochemistry (xylene + methanol) in the Clean Bench.

4. In the process of electrophoresis, sometimes the glue may drift and slip away. In many laboratories, students directly touch the glue by hand, which is very dangerous. For example, in the case of protein vertical electrophoresis, the voltage is high, so the human body can conduct electricity.

Through the observation of the operation in laboratories, we realize that it is very important to enhance the awareness of experimental safety before conducting experiments. Therefore, our team provides laboratory safety training for students who are working in the laboratories, so that they can develop correct experimental habits at the very beginning, rather than doing experiments blindly.

ii. We hand out Reagent Safety Manual in the school laboratories to strengthen the safety consciousness.

Toxic substances are commonly used in molecular biology laboratories.

1. The DMSO:
DMSO is dimethyl sulfoxide. DMSO has serious toxic effects and interacts with protein hydrophobic groups, resulting in protein denaturation, vascular toxicity and hepatorenal toxicity. DMSO is highly toxic and its volatilization should be noticed. If the skin is stained, it should be washed away with plenty of water and dilute 1%-5% ammonia. The commonest symptoms are nausea, vomiting, rashes and garlic, onion and oyster smell of the skin and exhaled gas. Inhalation: High volatile concentration may cause headache, dizziness, sedation, tingling, rashes and blisters. If dimethyl sulfoxide touches hydrated skin, there will be a thermal reaction.

2. EB (Ethidium Bromide):
Ethidium bromide is a highly sensitive fluorescent stain used to observe DNA in agarose and polyacrylamide gels.

Purification treatment of ethidium bromide solution: Because ethidium bromide has certain toxicity, after the end of the experiment, the solution containing EB should be purified and disposed of to avoid the environmental pollution and harm to human health. (1) For the solution with EB content greater than 0.5mg/ mL, it can be treated as follows: Dilute EB solution with water until the concentration is less than 0.5mg/ml; add 0.5mol/L KMnO4 to one volume, mix them entirely, then add the same amount of 25mol/L HCl, mix them entirely, and set them at room temperature for several hours; add one volume of 2.5mol/L NaOH, mix them and discard. (2) The solution with EB content less than 0.5mg/ml can be treated as follows: Add activated carbon in the amount of 1mg/ml, shake and mix it gently from time to time, and leave it at room temperature for one hour; (2) Filter it with filter paper and the activated carbon and filter paper after sealing discarded.

3. The DEPC:
DEPC, diethylprocarbonate, can inactivate various proteins and is a strong inhibitor of RNA enzyme. DEPC is a potential carcinogen. During operation, it should be carried out under the condition of ventilation as far as possible and should not touch the skin. DEPC is not very toxic, but the toxicity of inhalation is the strongest toxic. Operators need to wear masks when using it. If you accidentally get your hands on it, wash your hands immediately. RNaseAwayTM reagents can replace DEPC, and are easy to use, inexpensive and non-toxic.

4. Acrylamide:
It is a moderately toxic substance, and can be absorbed through the skin and respiratory tract into the human body. Therefore, when handling and using it, operators must wear protective equipment, such as protective clothing, respirator and protective gloves. Acrylamide is likely to cause neurotoxicity, as well as bringing about reproductive and developmental toxicity. Neurotoxic effect of peripheral nerve degenerative changes and function of the brain involved in learning, memory and other cognitive part of degenerative changes. Tests also show that acrylamide is a possible carcinogen. Besides, occupational exposure population epidemiological observation showed that the touch of low dose of acrylamide for a long time may cause sleepiness, change mood and memory and produce symptoms such as hallucinations and tremor, accompanied by peripheral neuropathy such as gloves sample feeling, sweating, and muscle weakness. Accumulated toxicity is not easy to detoxified. Patients with any of the following conditions can be listed as the subject for observation of chronic acrylamide poisoning: A. Part of skin exposed to acrylamide appears hyperhidrosis, damp and cold, peeling and erythema; B. Symptoms include limb numbness, tingling, weakness of the lower extremities and drowsiness; C. Neuro-emG showed suspicious neurogenic damage.

Treatment methods: B group vitamin, energy mixture, and complementary body therapy, physiotherapy and symptomatic treatment can be adopted. Support for treatment should also be strengthened in severe cases.

5. Nn-methylene diacrylamide is toxic and affects the central nervous system. Do not inhale powder.

6. DTT dithiothreitol, a strong reducing agent, gives off an unpleasant smell. If it is inhaled, swallowed or absorbed by the skin, it harms people. When using solid or high concentration storage fluids, operators should wear gloves and goggles and operate in a fume hood.

7. TEMED has strong neurotoxicity, prevents accidental suction, and is fast in operation and sealed when stored.

8. PMSF: Phenylmethyl sulfonyl fluoride [(PMSF), C7H7FO2S or C6H5CH2SO2F] is a highly toxic cholinesterase inhibitor. It is very destructive to the respiratory mucosa, eyes and skin. It can be fatal by inhalation, ingestion or skin absorption. Operators should wear proper gloves and safety glasses, and use them in a chemical fume hood. In case of contact, they should flush eyes or skin immediately with plenty of water. Discard contaminated work clothes.

9. Chloroform (CHCl3) irritates the skin, eyes, mucous membranes and respiratory tract. It is a carcinogen and can damage livers and kidneys. It is also volatile, so operators should avoid breathing in volatile gases. Operate with appropriate gloves and safety glasses, and in a chemical fume hood.

10. Formaldehyde (HCOH) is highly toxic and volatile, and is a carcinogen. It is easily absorbed through the skin and can irritate and damage the eyes, mucous membranes and upper respiratory tract. Avoid breathing in their vapors. Wear proper gloves and safety glasses. Operate in the chemical fume hood. Keep it away from heat, sparks and open flames.

11. Giemsa dyes can be fatal or cause blindness when ingested, and are toxic by inhalation and absorption through the skin. The potential danger is irreversible effects. Wear proper gloves and safety goggles. Operate in a chemical fume hood and do not inhale the powder.

12. Sodium azide (NaN3) is very toxic. It blocks the cytochrome electron transport system. Solutions containing sodium azide should be clearly marked. It may damage health by inhalation, ingestion or skin absorption. Wear proper gloves and safety goggles and be careful when handling it.

13. Sodium dodecyl sulfate (SDS) is a toxic irritant, and poses a risk to eyes. It possibly damage health by inhalation, ingestion or skin absorption. Wear proper gloves and safety goggles. Do not inhale the powder.

14. Trichloroacetic acid (TCA) is highly corrosive. Wear proper gloves and safety goggles.

15. Triton X-100 causes severe eye irritation and burns. Operators may suffer as a result of inhalation, ingestion or skin absorption. Wear proper gloves and goggles.

16. Ammonium persulfate [(NH4) 2S2O8] is extremely harmful to mucosal and upper respiratory tissues, eyes and skin. Inhaling it can be fatal. Wear appropriate gloves, safety glasses and protective clothing when operating. Operate in the fume hood and wash hands thoroughly after operation.

17. Trizol contains phenol toxic substances, such as skin contact Trizol. Rinse immediately with plenty of detergent and water. If you still feel unwell, consult your doctor. If only a small amount of exposure is treated and the symptoms are relieved, the problem may be minor.

18. Ultraviolet can damage the retina of the eye. Do not use naked eyes without protection. Ultraviolet light sources commonly used in laboratories include portable ultraviolet lamps and ultraviolet transmitters. It can only be seen through a filter or safety glass that absorbs harmful wavelengths. Ultraviolet light is also a mutagen and carcinogen. To minimize exposure, ensure that UV light sources are properly protected. Wear appropriate preventive gloves when operating under uv light.

In addition, we recognize some laboratory misconceptions about the new nucleic acid dye Goldview. There are still some companies on the market that claim Goldview is non-toxic and can replace EB, which misleads many experimenters to contact Goldview nucleic acid dye without taking protective measures. However, Goldview is acridine orange, also known as AO in legend. In the traditional cell apoptosis experiment, the dye used is acridine orange, also known as AO/EB staining test. EB cannot cross the intact cell membrane, while AO can cross the cell membrane to infect the nucleus, so as to distinguish apoptotic and non-apoptotic cells. What this experiment tells us is that since AO crosses cell membranes, AO is more toxic than EB. EB is a carcinogen, and Goldview is a lethal one. Cells that come into contact with Goldview die directly, while cells that come into contact with EB are severely injured. Goldview has been already on the list of carcinogens published by the World Health Organization's International Agency for Research on Cancer, although Goldview is a popular alternative to EB. It is highly undesirable to replace EB with Goldview in the laboratory, and to put laboratory staff at risk without explicitly informing them of its toxicity. To this end, our team investigated the use of nucleic acid dyes in laboratories of various colleges and universities (supplementary questionnaire, Figure P), and put forward suggestions to the laboratory management department of the university to promote the laboratory safety education. In the laboratory, we also made corresponding safety posters (supplementary drawings) to reduce the potential danger of the laboratory.

Public Awareness of Synthetic Biology

COVID-19 has made the public more concerned about biosafety. The Biosafety Law of the People's Republic of China was adopted at the 22nd session of the Standing Committee of the 13th National People's Congress on October 17, 2020, which reflects the rising status of biosafety in the eyes of the state and the public. The outbreak of microbiological experiment in the public opinion has a negative impact, will the public laboratory waste water and waste pollution living security concerns to be reckoned with. To this end, our team try to deal with waste liquid and waste sewage treatment center, and visit the school and live sharing the experiment process, to eliminate the public concern about our experiment.

Description: https://2020.igem.org/Team:FAFU-CHINA/Description

Attributions: https://2020.igem.org/Team:FAFU-CHINA/Attributions

Our goals：

1. Promote the cultivation of fungus grass, hoping that people all over the world can profit from Juncao.

2. Designed a universal integrated fermentation system to improve the current situation of the biological treatment industry;Without relying too much on the reaction apparatus, the bioethanol can be converted into one time only by using ordinary fermentation tanks.

3. It can reduce or completely eliminate the cost of enzyme extraction, purification, preservation and transportation.The selected chassis has a high biosafety, after full research, has been widely used in industrial production.

4. Devote to the design of high-temperature fermentation system, because high-temperature conditions can improve the reaction speed and avoid the contamination of the system by exotic bacteria (which can reduce the cost of using antibiotics and preservatives).

5. Compared with the traditional fermentation industry, it relies on the chassis biology to produce enzymes, which are directly secreted into the fermentation tank, avoiding the cumbersome intermediate process.

Juncao, native to Africa, has the characteristics of high luminous efficiency, high water utilization rate, developed roots, good water retention and soil consolidation, and strong stress resistance.

In terms of biosafety, studies have shown that ^[1] planting Juncao will not cause biological invasion.So after a little assessment based on the environment of the introduced area, we can determine whether the Juncao is suitable for introduction in the area.

Economic benefits

Its advantage: Juncao as C4 plants, its photosynthetic conversion rate is 6-21 times that of broadleaf trees, using it as biomass fuel for power generation, can achieve zero carbon dioxide emissions.The dry heat value of mycelia was 1.15 times that of maize, and the biomass fuel characteristics were better than maize.And its ash content is only half of corn, convenient for slag disposal.Juncao have low sulfur content and burn less sulfur.Therefore, the raw material for power generation using Juncao biomass is relatively environmentally friendly ^[2].

Ecological benefits

Juncao can reduce soil pH and total potassium and increase organic matter, total nitrogen, total phosphorus, bacteria, fungi, actinomycetes, catalase, polyphenol oxidase and urease.To eliminate the influence of bare land, the pH value and total potassium of the soil were reduced by 3.5% and 4.6%, respectively, and the organic matter, total nitrogen and fungi were increased by more than 100%, significantly improving the soil environment ^[3] .

Giant Juncao root developed, can improve the soil particle structure.In addition, the leaves of giant Juncao and other plant residues increase soil humus, forming mineral nutrients under the action of microorganisms and gradually leaching into the soil, which can improve soil organic matter and supplement soil nutrition.

This is similar to the effect of crop residues, which fertilize the soil and preserve and increase soil organic matter.Studies have shown [4] that crop residues significantly affect soil organic matter composition, improve soil porosity, improve soil permeability, water and fertilizer retention, and enhance soil buffering after being applied into the soil.

In summary, the planting of megalosus had a promoting effect on the improvement of soil microbial quantity and soil enzyme activity, indicating that the planting of megalosus had a positive effect on the improvement of soil quality.At the same time, the changes of soil fertility of megalosus with different growth years indicated that planting megalosus could improve soil fertility and change the structure of soil microbial community.

Therefore, the development of Juncao as an energy crop can achieve ecological and economic win-win results.

Social benefits

In addition, a series of international cooperation has been carried out based on the introduction of Juncao.In Papua New Guinea, Fiji, Rwanda and other countries, the United Nations 2030 Agenda for Sustainable Development Fungus Technology Demonstration Base has been established.The industrialization of Juncao can increase employment, eliminate poverty, reduce hunger, use renewable energy, deal with climate change, help developing countries solve development problems, and implement the United Nations 2030 Sustainable Development

Biodegradation of lignocellulosic raw materials is a complex process with many influencing factors, including the types of engineering bacteria, processing conditions, the optimal conditions and mechanism of action of various lignocellulosic enzymes. Due to shortcomings such as time-consuming and high cost, its large-scale industrial application is restricted.

We hope to improve this problem by designing an integrated fermentation system. In order to design a suitable integrated fermentation system, we have to understand the composition and degradation principle of lignocellulose, the action mechanism of lignocellulase, the minimum combination of lignocellulase and the action mechanism of cellulosomes.

To use lignocellulose, it is necessary to degrade lignin, hemicellulose and cellulose in sequence. By consulting the literature, we have collected the smallest combination of enzymes that degrade lignin; by using the uni-port database, we have found a batch of isozymes with similar optimal pH and temperature. The distribution of enzymes from different sources to different chassis organisms is the basis of our co-cultivation system.

In addition, we have considered biosafety issues very seriously. The chassis organisms we use are Escherichia coli and yeast, which are highly safe organisms. The entire fermentation process is carried out in a sealed container. After the reaction, the process of high-temperature concentration of the ethanol mother liquor will kill all organisms in the bottom plate, and all members of the experimental group have received laboratory safety training before entering the laboratory.

The concept of an integrated fermentation system has been adjusted based on rationality and cost factors. We especially want to thank our cooperation team BNU-China, who have put forward many constructive suggestions for our system design and system simplification.

Our ultimate goal:

1. Promote the cultivation of Juncao, hoping that farmers all over the world can profit from planting Juncao.

2. Design a universal integrated fermentation system to improve the current status of the biological treatment industry; do not rely too much on the reaction equipment, as long as the ordinary fermentation tank is used to achieve the one-time conversion of bioethanol.

(Develop Juncao industry to benefit all mankind.)

IDEA

Our project was inspired by the 2019SDU-China project. Their wiki introduces the advantages and potential of the co-cultivation system for the production of bioenergy, and they have successfully built a light-controlled co-cultivation system. We also hope to use lignocellulose to produce bioethanol by designing a co-cultivation system.

In the back hills of our campus, there are many Juncao plants. We know its excellent properties, such as high biomass content, fast growth, no biological invasion, strong resistance to stress, and easy fermentation, so we want to use Juncao As a biomass raw material for an integrated fermentation system.

We got in touch with Li zhao long, the captain of 2019SDU-China. We discussed with him on the design of the co-cultivation system, he put forward a lot of valuable opinions and provided us with the B(3) plasmid used in their co-cultivation system. Through molecular cloning technology, we transformed it into the plasmid used in our co-culture system.

Juncao has been used in the industrial production of biogas, and the comprehensive properties of Juncao are significantly better than corn, so we have enough reasons to believe that Juncao can be used for industrial production of bioethanol. The development of Juncao with ecological, economic and social benefits into energy crops has been unanimously recognized by team members and instructors, so we have determined this year's topic.

Co-culture system

Combined bioprocessing is the integration of cellulose secretion, cellulose hydrolysis and ethanol fermentation into one step, directly using cellulose micro-carbon sources to produce ethanol, which is a more economical and convenient process in biofuel production. We have used many co-cultivation systems for reference, and we hope to complete the following four tasks by designing a co-cultivation system of E. coli and yeast: the degradation of lignin, the degradation of hemicellulose, the degradation of cellulose, and the fermentation of ethanol.

Co-cultivation of Saccharomyces cerevisiae and Escherichia coli to produce naringin

Naringin is a valuable natural product. Xylose creates harmonious culture conditions for the two engineered microorganisms. It is the only non-competitive carbon source and does not produce secondary ethanol.

Yeast co-culture system

The system contains four engineered yeast strains. One type of yeast displays scaffold protein on its surface. The scaffold protein contains three different cohesin domains. The other three strains secrete cellulase with the corresponding dockerin tag. Based on the specific interaction between cohesin protein and docking protein, cellulase can be assembled on the cell surface to form cellulosome. By adjusting the ratio of strains, cellulosome assembly, cellulose hydrolysis, and ethanol production can be optimized. The final ethanol output can reach 1.87 g/L, which is 93% of the theoretical maximum yield.

Our integrated fermentation system

Based on the principles and advantages of the above co-cultivation system, we designed the following integrated fermentation system.

Our idea is to design a co-cultivation system composed of 3 kinds of chassis organisms. The entire system can be self-regulated depending on the dynamic changes of the initial substrate and intermediate product concentration to achieve the optimal allocation of biomass energy to the system.

Composition and degradation of lignocellulose

Lignocellulose mainly includes cellulose, hemicellulose and lignin

The main components of lignocellulose are cross-linked with each other to form a dense natural cell wall structure. This complex and stable three-dimensional structure forms a natural anti-degradation barrier. Therefore, it is difficult to directly degrade lignocellulose by enzymes or microorganisms under normal circumstances. This undoubtedly makes the efficient conversion process of lignocellulose to fuel ethanol extremely difficult.

In industrial production, the chemical pretreatment method is not only costly, but the chemical reagents used are also easy to pollute the environment. Although the physical pretreatment method, especially the steam explosion method, has circumvented the problems of the chemical pretreatment method to a certain extent, its production cost is still high. Through the development of biological pretreatment technology, it is hoped that biomass raw materials can be treated efficiently at low cost and will not cause adverse effects on the environment.

First, the outermost layer of lignin is degraded; then, the hemicellulose wrapped around the cellulose is degraded; and finally, the cellulose that contains the highest content of lignocellulose is degraded.

According to the reaction principle of Fenton reaction, we have found two enzymes. One is aromatic alcohol oxidase, which can produce hydrogen peroxide; the other is horseradish peroxidase, which consumes hydrogen peroxide and generates free radicals to attack lignin.

The composition of Juncao hemicellulose is simple, mainly xylan, which can be degraded synergistically with cellulose.

Degradation of cellulose

Classic cellulase: celccA （endocellulose）, celccE （exonuclease）, Ccel-2454 （β-glucosidase）. They act on the non-crystalline area of cellulose.

New cellulase: LPMO (lysocellulose monooxygenase), CDH (cellobiose dehydrogenase). They act on the crystallization area of cellulase. （At the same time, they can also act on lignin. )

The LPMO is shaped like a triangular pyramid, and the catalytic active center is located on its flat bottom surface. This structure allows the LPMO to be closely attached to the surface of the substrate. (This allows LPMO to degrade certain dense materials)

1. Promote the cultivation of fungus grass, hoping that people all over the world can profit from Juncao.

2. Designed a universal integrated fermentation system to improve the current situation of the biological treatment industry;Without relying too much on the reaction apparatus, the bioethanol can be converted into one time only by using ordinary fermentation tanks.

3. It can reduce or completely eliminate the cost of enzyme extraction, purification, preservation and transportation.The selected chassis has a high biosafety, after full research, has been widely used in industrial production.

4. Devote to the design of high-temperature fermentation system, because high-temperature conditions can improve the reaction speed and avoid the contamination of the system by exotic bacteria (which can reduce the cost of using antibiotics and preservatives).

5. Compared with the traditional fermentation industry, it relies on the chassis biology to produce enzymes, which are directly secreted into the fermentation tank, avoiding the cumbersome intermediate process.

Chimeric enzyme:The simplest Chimeric enzymeis to add a docking protein domain on the basis of the original enzyme. Allow it to bind to the scaffold protein with the corresponding cohesin protein domain.

In addition, other functional domains and some tags can be added to the Chimeric enzyme.

We designed the composite element BBa_K3550012 to be added to the C-terminus of the enzyme, It can convert the reaction form of free enzymes so that it can be added to cellosome.^[8]

BBa_K3550012 contains a linker, a dockerin and a protein purification tag with TEV. Generally, it can be directly added to the C-terminal of the DNA sequence of lignocellulase. In particular, the link of BBa_K3550012 in our wiki is incorrectly linked to BBa_K3550002, so we have added the basic information of BBa_K3550012 in the poster.

Construction of cellulosome:The cellulosomes have a very orderly structure and have high cellulose degradation efficiency. It specifically includes a complex enzyme with a docking protein domain, a secondary scaffold protein with an cohesin protein domain, and a scaffold protein with a lectin subunit (a domain for anchoring on a cell).

By designing and combining scaffold proteins, we can design cellulosome with larger scale and more functions.

On the other hand, we can directionally modify hemicellulase and ligninase so that they can be added to cellulosome.

Verification of self-assembly of biological macromolecules:cellulosome的组装率。The assembly rate of cellulosome can be verified by giving different fluorescent signals to different components and observing through laser confocal microscope.

Theoretically speaking, the successful construction of large-scale cellulosome means that we have designed a super enzyme that can degrade lignocellulose, which will help realize the one-time conversion of bioenergy from raw materials to products.

Lignin determination model

Obtain the lignin content in different environments, obtain the constant of glucose conversion to cellulose content, and obtain the standard curve diagram of lignin content.

Logistic regression model

The growth curve diagram can be obtained for future estimation of the number.The growth rate can be obtained, and the growth curve equation can be obtained. Logistic growth model is established:

The solution is

Response surface method optimization model

Obtain the weight of different variable factors on the experimental results, which influence value is greater.

Industrial Modeling

We built multiple ideal bioreacter model. For example,we use the M-M equation to find the maximum reaction rate, and use the substrate consumption model to calculate mass preservation.Monod equations are used to describe the relationship between cell-specific growth rates and substrate concentrations. Arrhennius equations are used to determine quantitative relationships between specific growth rates and temperatures, and Luedeking-Piret equations are used to describe coupling relationships.

Model 1 Lignin determination model

Formula.：

Model 2 Logistic Model

Formula：

Model 3 Response surface method optimization model

Configure the optimal medium for cellulase-producing bacteria BL21 use Box-Behnken design, perform response surface analysis, and use Design Expert software to perform statistical analysis on the results of filter paper enzyme activity to obtain multiple linear regression equations.

Model 4 Industrial Modeling

Model 4.1: Mass scale equation

The time required for the reaction concentration CS conversion to CS0 can be calculated

Model 4.2: Equation M-M

Find the maximum reaction rate of the enzyme

Model 4.3: Yield coefficient

The overall yield coefficient/instantaneous rate factor

Theoretical yield coefficient/esoolysed rate coefficient

Model 4.4: Mass accounting of the substrate consumption model

When calculating the rate at which the substrate is consumed, the cell's products are produced at the fastest rate

Model 4.5: The general form of the black box metering model

Calculate the yield factor

Model 4.6: Monod equation

The relationship between the growth rate of the indicated cell than the concentration of the substrate

Model 4.7: If you consider fixing it according to the suppressed model

The purpose of the correction is to determine the substrate concentration at the maximum growth rate

Model 4.8: Effect of temperature on maximum growth rate

Model 4.9: Luedeking-Piret equation

Represents product generation rate (coupled and unsymlinked)

Model 4.10: 2-phase enzyme reaction

Assuming a certain substrate conversion rate, the reaction time is related to the substrate concentration, the Michaelis constant, and the maximum reaction rate

Model 4.11: Reaction time required for growth and decelerator

Model 4.12: Optimal reaction time

In order to make the industrial production more perfect and the cost process easier. In the industrial modeling part of iGEM, we simulate the lignin measurement experiment based on the use of experimental data and draw a linear fitting standard curve, and then use the logistic model to fit the E. coli BL21 strain multiple times to obtain the E. coli growth curve. And how to configure the best culture medium for the cellulase-producing bacteria BL21 and how to use the response surface method to draw conclusions to further improvement.In order to make its industrial application effect better, we have established from multiple perspectives Multiple models, using the ideal bioreactor model, and using the M-M equation to determine the maximum reaction rate, and calculated according to the actual situation. Most importantly, we have also optimized the above models, such as using Aiba equation, Yano equation and Levenspiel equation to modify the model, etc. This greatly improves the accuracy and efficiency of the industry to a certain extent. In addition to the reduction of cost value, it can also reduce the impact on the environment to a certain extent, which is very conducive to the long-term development of industrial production.

When our experiments have preliminary results, we can bring them in those models and work out the values of parameters that can be optimized. At the same time, we can set a series of gradient control experiments based on these parameters for further optimization.

The energy problem is becoming increasingly prominent in the global problem, we are looking for new energy elements to solve the problem. Our FAFU-CHINA team focuses on lignocellulose. In order to better understand cellulose, we have carried out a variety of cooperation.

Partnership

In order to better understand the experiments of other teams,but also to make our experiments better, our team is actively looking for partners.

We actively cooperate with other universities in the course of the experiment. We discussed the experiment with BNU-China, and we put forward a lot of valuable suggestions on their experiment, such as we suggested that they use a new type of test strip to detect foodborne pathogens. They also suggested that we could use yeast to make wine, and we could also produce bioethanol, and we attended a live lecture in Beijing at their invitation, during which our experimental project was further popularized. at the same time, it has also greatly deepened the close ties between our two teams.

Collaboration

In the course of the experiment, the team actively cooperated with other teams and successfully solved many problems and puzzles in the process of the experiment.

Factory

The combination of team experiments and human practice is very important, so we actively carry out offline practice, visit and investigate relevant factories, and we benefit a lot from this process.

Eastern China iGEM Meetup

25 teams discussed together, and The CEO of Beijing Zeno Technology, Zhu Tianze, and Hassnain gave advice.

Northeastern coastal areas online meetup

iGEM co-education

Five universities jointly carried out the "public activity of popularizing synthetic biology", with more than 30,000 participants.

In mid August, together with thuigem, PKU iGEM, cauigem and BNU iGEM, we launched a week-long "science popularization public activity of synthetic biology" online. The teaching included six aspects: synthetic biology foundation, medical treatment, agricultural food, energy, information art and industrial environment.

CCIC

On August 29, solstice, August 31, 2020, our team participated in the 7th CCiC (China iGEMer community meeting). A total of 48 teams participated in CCiC2020, and all of our teammates attended the meeting.

Green environmental protection

In the desert area to promote the cultivation of Juncao.

In recent years, Professor Lin and his team have shown that "mycelia" can be an effective species for controlling desertification.

In the future, we plan to cooperate with Alibaba's ant Forest and the China Greening Foundation to cultivate fungus, combat desertification and produce bioenergy. Since 2017, Ant Financial Has donated funds to the China Green Foundation,Set up the "Ant Forest" public welfare project.

We believe that the 1+1>2 effect can be produced! Our projects can not only prevent and control desertification and produce bioenergy, but also enable the public to understand synthetic biology and experience richer environmental experience.

NEFU-China

Jilin_China

Jiangnan

Intergrated Human Practices

To solve our problems.We can just talk to Director.Hua chaung Wu online.We also interviewed Prof. Bin Liu,Prof. Zhanxi Lin, Prof.Zhang Xingtan.They all gave us a lot of advice, a lot of help with synthetic biology and projects.

The complex spatial structure of lignin not only hinders the enzyme degradation of cellulose and hemicellulose in lignocellulosic raw materials, but also produces non-productive adsorption of hydrolytic enzymes, which seriously affects the efficiency of substrate enzymatic hydrolysis, so we hope that Separate the degradation of lignin, cellulose and hemicellulose. Through the means of inducing expression, we can achieve this goal.

The cost of light regulation is obviously lower than that of chemical induction, but chemical induction is more stable in terms of effect. In order to better separate the degradation of lignin, cellulose, and hemicellulose, we finally chose the chemical induction method. We expect that the effect of the chemical induction method is to induce the expression of ligninase and inhibit the expression of cellulose and hemicellulase when lactose is present; when the lactose is depleted, the expression of ligninase stops and the cellulase and hemicellulase are relieved. Due to the extremely limited time and funding this year, we only used reporter genes (gfp, rfp) to initially verify the function of the system.

The picture above shows the 2019-SDU co-cultivation system. Different strains can emit different colors of fluorescence.

We hope that when the inducer lactose is present, only red fluorescence exists in our co-culture system; when the inducer is exhausted, only green fluorescence exists in the co-culture system. During the entire culture process, the red and green fluorescence coexist as short as possible, in order to preliminarily simulate the function of the dual-plasmid system to separate the degradation of lignin from the degradation of cellulose and hemicellulose.

If time permits, we will first verify the functions of the two plasmids separately. When lactose is present, Pet28b(+)-Gfp does not fluoresce green; when lactose is not present, Pet28b(+)-Gfp fluoresces green; when lactose is present, Pet28b(+)-Rfp fluoresces red; when lactose is not present, Pet28b (+)-Rfp does not fluoresce red. We will set up several experimental groups with different concentrations of inducer, and then co-culture the strains with the two plasmids, observe the samples through a laser confocal microscope regularly, and find the optimal inducer concentration based on the overlap time of the two-color fluorescence.

Pick the fungus

In order to better prove the effect of the artificial fermentation system, we plan to compare the ability of degrading cellulose with wild-type cellulase-producing strains.

We took rotten soil at school, and isolated strains capable of degrading cellulose from the soil. In total, we isolated 11 strains of bacteria from the soil, and performed evolutionary tree analysis, among which 7 strains were identified as their species

FZ-FAFU-1（Acetobacter sp. KF484802.1）

FZ-FAFU-3（Aeromonas sp.KY971270.1:13-1044）

FZ-FAFU-4（Bacterium , THCL7EU086553.1:255-547）

FZ-FAFU-7（Enterobacteriaceae sp. MH135803.2:2-706）

FZ-FAFU-8（Vibrio parahaem , MG593213.1:192-374）

FZ-FAFU-9（Bacillus cereus , MK829512.1:2-1011）

FZ-FAFU-11（Acinetobacter sp. GU272398.1:1-952）

DNS

DNS was used to determine how much reducing sugar the strain produced in the CMC-NA medium in the survival state compared with the death state, so as to calculate the decomposability of the strain.

soil samples

We took rotten soil near the School's Zhonghua Garden and sugarcane base as soil samples for the separation of wild bacteria capable of cellulose degradation.

We counted a total of about 12 plates, two days after the culture of bacterial growth, more than half of the plate culture medium bacteria growth is good, suitable for further experiments.

Plan A

After our first discussion, we decided to select the single colony into the EP tube containing 1ml CMC-Na liquid medium. After the liquid in the EP tube becomes cloudy, we want to dip the bacteria liquid into the middle of the Congo red medium plate with a filter paper. If there is a hydrolytic ring, it will be the desired strain. The results showed that the growth rate was extremely slow, and there was still no growth two days later (the bacterial fluid became cloudy 7 days later).

Plan B

So we decided to use the second method, we used a pipette to pick the more obvious single colony on the medium, and transferred to the new solid screening medium plate (CMC-Na), two plates for every single colony.

The picture shows the colony morphology of the strains we picked.

Then we prepared 0.2% Congo red staining reagent. We added 1ml Congo red reagent directly to the plate with bacterial colonies and let it stand for half an hour. After that, the Congo red reagent was poured out, and the plate was cleaned several times with double steaming water, and the hydrolytic ring was carefully observed.

We took 400ul from each of them and sent them to the company for sequencing, using 27F, 1492R for sequencing.、

phylogenetic tree

After getting the sequence, we use the Maximum Likelihood Method (ML) to construct a phylogenetic tree, which is a method based on nonlinear spectrum estimation; calculate the likelihood function for all possible phylogenetic trees, and then select the one with the largest likelihood function value The phylogenetic tree model is the best choice.

As shown in the figure, we measured the 16S_rDNA sequence of the bacteria with 27F and 1492R primers to identify the different species of bacteria and divide them into six subclasses I, II, III, IV, V, VI. According to the results of phylogenetic tree cluster classification, it can be inferred that they may have a common ancestor.

The numbers on the branches mean the reliability percent of Bootstraps value basedon1000replication; The branches of different classes were painted with different colour（Red branches represent the first category, black branches represent the second category, golden branches represent the third category, green branches represent the fourth category, grey branches represent the fifth category, and blue branches represent the Sixth category）, Different colours and symbols represent different species, and each arc of different colours represents a different subclass (Class I~Class VI).

FZ-FAFU-1（Acetobacter sp. KF484802.1）

FZ-FAFU-3（Aeromonas sp.KY971270.1:13-1044）

FZ-FAFU-4（Bacterium , THCL7EU086553.1:255-547）

FZ-FAFU-7（Enterobacteriaceae sp. MH135803.2:2-706）

FZ-FAFU-8（Vibrio parahaem , MG593213.1:192-374）

FZ-FAFU-9（Bacillus cereus , MK829512.1:2-1011）

FZ-FAFU-11（Acinetobacter sp. GU272398.1:1-952）

Glucose as standard

We first prepare DNS solution, and then use the newly prepared DNS solution as the standard glucose curve.

Glucose standard solution was prepared. First, 5-10 g of glucose was weighed and dried to constant weight at 60℃ for about 3-5 days. Then, 1g of glucose was weighed and dried to constant weight to prepare reserve solution (concentration: 1mg/mL) : 0.1g of glucose was stabilized to 100ml.

The six tubes were then bathed in boiling water for 15 minutes, rapidly cooled and stabilized to 25 mL. Under the condition of λ= 540 nm, the absorbance value was measured with an Ultraviolet spectrophotometer. The glucose concentration was used as the x-coordinate and the measured absorbance value as the y-coordinate to draw the standard curve of glucose.

DNS

We selected the bacteria into CMC-Na medium, put them into a shaker at 28℃ for culture at 180R /min for 3-4 days, and then took 1mL bacterial solution into an EP tube, centrifuged at 8000rpm for 5 min, and took the supernatant as the crude enzyme solution. We added 50uL supernatant to 150uL 1% cellulose solution as the experimental group. The supernatant solution of 150uL 1% cellulose was added to 50uL boiling water for 30 min as the control group. We put the experimental group and the control group into the water bath to keep the temperature at 50℃, and the gradient was 5min, 10min, 15min, and 30min. After adding 150uL DNS and 100 L NaOH solution, boiling water bath for 5min. Finally, the volume was fixed to 1 mL. After zeroing in the blank group, the OD540 value was measured and the average value was taken.

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