Introduction to the project

Energy depletion of the earth has been a critical problem. We must find another fuels to replace petroleum. The most common biomass fuel is ethanol. But ethanol still can not be substituted completely for petroleum, because of its causticity to the metal of engine. If we want to generate energy with ethanol only, we may have to change whole engine to be in different material. That’s very inconvenient. Unlike ethanol, isobutanol can be used on car engine safely. In fact, isobutanol has been used in many purposes. Here comes a lot of applications and advantages of isobutanol. For example, organic solvent, reactant of organic mechanism, antifreeze and so on. One of the advantages is that its high ratio of the heat of combustion is very close to petroleum. It means that isobutanol is suitable to be fuels. So we finally choose it to be our project. What’s more, if we can generate energy from cellulose by converting it into glucose or even isobutanol, it will be more helpful to people’s life. We believed that isobutanol must have wide development in the future. However, according to many research, we found that the E.coli used for producing isobutanol always died for the rising concentration of isobutanol, so the yield always couldn’t get higher. Fortunately, we figured out two innovative and brilliant solutions to solve this serious problem. We will introduce our idea on our wiki in detail.

　Project details

　Enzyme for isobutanol

To produce isobutanol, we use four enzymes to caltalyze Pyruvate. We cloned the genes which can be translated into the enzymes─ AlsS, ilvC, ilvD, KivD, adjusted the expression of the genes, and made sure every intermediates can be catalyzed by every next enzyme. The overall pathway is shown in Figure 1. As glucose can be catalyzed into pyruvate by Glycolysis, we choose glucose as the resource in biosynthetic pathway. Then, pyruvate will be turned into isobutanol by the enzymes shown in Figure 2.

(Figure 1)

(Figure 2)

　Temperature control system

The low temperature release system is a way to let e.coli produce isobutanol efficiently . Because isobutanol and isobutyaldehyde are toxic to the e.coli , the system avoid e.coli facing them at the beginning . The following picture is our system.

At the beginning , we will let E.coli stay in 37°C environment. After having enough 2-Ketoisovalerate , we will move E.coli into 30°C environment for producing the final product , isobutanol. It can make us get isobutanol successfully and efficiently.

This is our biobrick. The most important part of our biobrick is 37°C ribosome binding site gene. We separate our biobrick into two parts . The first part is which has 37℃ ribosome binding site gene and the second part is under the first one.

And now we're introducing how our system works.

When being in 37°C environment, the first part will be translated and produce tetR protein to inhibit Ptet promoter. The second part will not be translated. Then we can produce intermediate , 2-Ketoisovalerate.

After getting enough 2-Ketoisovalerate , E.coli will stay in 30°C environment. The ribosome will not bind the 37°C ribosome binding site and tetR genes will not be translated. Then the second part will be translated successfully. At the end , we can get the isobutanol.

　Result

1. The preparing tests for our project

Figure 1. Activate our E.coli overnight. Then, transfer it into the new medium with the microaerobic environment until O.D. reaching 0.2. After that, test the O.D. value every 4 hours.

We did an experiment to prove the isobutanol is truly toxic to the E.coli. The data shows that the higher concentration of the isobutanol was in the medium, the lower O.D value could be obtained.

Figure 2. Culture DH5a, DH10B, JM109, MG1655,EPI300 strains in the 3.6% glucose M9 medium and 37℃ environment until O.D. reaching 0.2. Then, continue culturing it for 24 hours and measure the production of isobutanol.

First, we tested how much isobutanol would be produced by the five kinds of strains in our low temperature release system. According to the result, we knew that the DH5α was the strain which produced the most of isobutanol. So, we chose the DH5α as the main strain producing isobutanol in our project.

Figure 3. Incubate the DH5α strain in the common medium, M9, and other 2 kinds of mediums, M9T(M9+ trace metal mix) and M9TY(M9+ trace metal mix+ yeast extract).

In addition, we cultured the DH5α strain in the common medium, M9, and other 2 kinds of mediums to see which medium was the best for it to produce isobutanol in the low temperature release system. In this data, we found that M9TY medium was the most appropriate medium for DH5α strain. As a result, we decided to use the M9TY medium.

Figure 4. Culture our E.coli in the 37゜C environment which is appropriate for E.coli growing at the beginning. Then, change the temperature after being 0 hour or 4 hours or other hours.

This is our main idea to design the experiment of the temperature control system. It is that we cultured our E.coli in the 37゜C environment which is appropriate for E.coli growing at the beginning. Then, we would change the temperature after being 0 hr or 4hrs or other times showed by the figure 4.

(1)GC analysis of the isobutanol production-turning from 37゜C environment into 32゜C environment sample

Figure 5. Culture our E.coli in the 37゜C environment at the beginning. Then, change the environment into 32゜C after being 0 hour, 12 hours , 16 hours, 20 hours and 24 hours.

The report indicates that changing from the 37゜C environment into the lower temperature environment did truly get more isobutanol. We could see that E.coli being in 37゜C environment for 20 hours and in 32゜C environment for 4 hours have the highest % concentration of isobutanol.

Figure 6. Mark the second circuit with the fluorescent protein to test the expression of kivD enzyme.

We also used the fluorescent protein to mark the second circuit of our biobrick. The data tells us that kivD enzyme being in the 37゜C environment had the lower expression than being in the 30゜C environment and the 25゜C environment.

All of the reports mean that our low temperature release system does truly work!

(2)GC analysis of the isobutanol production-turning from 37゜C environment into 42゜C environment sample

Figure 7. Culture our E.coli in the 37゜C environment at the beginning. Then, change the environment into 42゜C after being 0 hour, 12 hours , 16 hours, 20 hours and 24 hours.

According to the data, we discovered that E.coli being in 42゜C environment for 24 hours would have higher % concentration of isobutanol than being in 37゜C environment at the beginning. It had the high reproducibility and was really surprising to all of us because it was out of our expectation ! We pondered the possible reason might that the kivD enzyme being in the 37゜C environment had lower activity than being in the 42゜C environment. In addition, there was lack of toxic byproducts because of low expression of kivD enzyme in the 42゜C environment.

Figure 8. Culture our DH5α strain in the medium of 3.6% glucose or 5% glycerol for three days. Test the sample by GC every 24 hours.

After learning that glycerol is the redundant product of the petroleum pyrolysis, we wanted to reduce the useless byproduct of petroleum Industry on earth. We did another experiment to see whether glycerol was possible to be our E.coli ‘s ingredient or not. We discovered that using glycerol as the ingredient could get the 1/3 of the yield of isobutanol produced by using glucose as the ingredient. We also observed that the % concentration of isobutanol being in the 42゜C environment was the most highest in the three kinds of temperature environments. We proved the reproducibility of it and considered that we might optimize our temperature control system according to these reports.

　Zinc finger

Zinc finger proteins contain a DNA binding domain and a functional domain. It could recognize specific DNA sequence, which named DNA program. Zinc fingers could tightly bind to specific DNA or RNA sequence. With this feature, we expected to build a production line to help us make isobutanol. We replace the functional domain with our enzymes to create fusion protein. The enzymes disperse around the cell; therefore the productivity of isobutanol will be low.

We put the enzymes in order. When the intermediates are produced, it could have the next reaction as quickly as possible. The final product, isobutyraldehyde will be converted into isobutanol by ADH in E.coli.

(Point mutation)

We found that there are only five nucleotides between the genes, HIVC and ilvD after ligating. (ATG are the first three nucleotides of the ilvD gene.) According to the triplet nature of gene expression by codons, it would cause a frameshift mutation, which cause the reading of the condons code for incorrect amino acid.

(1)So we inserted an A into the site after the HIVC gene. As a result, the sequence between the two genes accomplishes six nucleotides.

(2)However, we found a stop codon, TAG in every connection between the zinc fingers and the enzymes that surprised us.

(3)We made a point mutation again to change the fifth nucleotide from A to T. Therefore; the stop codon (TAG) is changed to TTG codes for leucine.

　Result

(developing)

　Instrument

(1)37°C

At the first step, we put the e.coli we designed and M9 medium ,containing 36 g/L glucose, 5 g/L yeast extract,100 μg/ml ampicillin, 30 μg/ml kanamycin, and 1,000th dilution of Trace Metal Mix A5 (2.86 g H3BO3, 1.81 g MnCl2 ⋅4H2O, 0.222 g ZnSO4 ⋅7H2O, 0.39 g Na2MoO4⋅2H2O, 0.079 g CuSO4⋅5H2O, 49.4 mg Co(NO3)2⋅6H2O per liter water) ,into the first tank. Then, we culture the e.coli in the 37°C environment for three hours which means that we put the tank in the warm bath to let e.coli produce the intermediate,2-ketoisovalerate.

(2)30°C

Afterward, we put our E.coli into 30°C environment maintained by warm bath for 3 days incubation. Our low temperature system would initiate expressing of kivd which would convert 2-ketoisovalerate to isobutyraldehyde. Then, isobutyraldehyde would be converted into isobutanol by E.coli's own ADH.

(3) preliminary distillation

After incubating the “E.coline” in the 30°C environment for three days, the concentration of isobutanol is high enough to be collected. We prepared two flasks which half-filled cold water and each of them was equipped with a condenser. The three flasks were linked with pipes. One end of the pipe (air out) must be under the water level, so that the air would expose into water of the destined flask. We pump air to strip the isobutanol to the flask for product collection. If isobutanol could be transferred from the fermentation flask, we expected the production rate could extend tremendously and the following condensate collector will obtain higher concentration of isobutanol than the previous fermentation flask. By having this higher concentrated isobutanol, isobutanol purification will be much more favorable to be conducted.

　Conclusion

The core aim of our “E.coline” project is to generate isobutanol, a promising eco-fuel, in a productive and efficient way.

To produce isobutanol, We first put four pyruvate catalytic enzyme genes: AlsS, ilvC, ilvD, KivD all together. And we then designed a temperature control system to avoid e.coli from being polluted by isobutyaldehyde. According to our data, our temperature control system is proved to work great.

And to produce isobutanol more efficiently, we combine the zinc fingers and our enzymes together and rank the fusion proteins in the catalytic pathway order. We have also mutated the stop codons in our fusion proteins.

　Optimization

(developing)

　Future works

In order to realize our idea to change trash into fuel, we did some research. Therefore, the first thing we have to do is figure out how to degrade the cellulose. First, we want to get xylose from cellulose through xylanase. Xylanase is a class of enzyme which degrades the linear polysaccharide beta-1,4-xylan into xylose, thus breaks down hemicellulose, one of the major components of plant cell walls. Xulose is a good carbon source. As such, xylanase plays a major role in micro-organisms thriving on plant sources (mammals, conversely, do not produce xylanase).

According to the Journal of Applied Microbiology ( Y.P. Chen et al. 2011), the cell-surface display of Cex, which encodes xylanase from Cellulomonas fimi, was constructed on E. coli using PgsA as the anchor protein. Through the graph from this paper, it shows that Cex do have the activity to catalyze xylan into xylose.

Another benefit of using PgsA fusion enzyme is that it can lead isobutanol-producing enzymes catalyze through consolidated bioprocessing(CBP). the CBP in converting Cellulose into isobutanol requires combinations of biological events(production of xylanases, hydrolysis of the polysaccharides in the biomass, temperature controlling, and production of isobutanol) in one reactor. CBP has gained recognition as a potential breakthrough for low-cost biomass processing. So, if we incubate E. coli with this mechanism with our isobutanol-synthesis E. coli, we can cost down the expenses of enzyme purification. Finally, the reactor as a whole will be more like a biofuel production line!

In the future, we will find out the reaction time of every step. For example, how long does it take for producing a certain concentration of 2-ketoisovalerate per 300 ml culture medium? With the data, we can decide the Incubation time and the flow to the next flask totally controlled by the pump. Thus, we can build an automatic instrument to produce isobutanol inexhaustibly.

We use the above introduced cellulase to produce glucose as ingredient in the first tank(preparation). The biosynthetic production of isobutanol based on our project (R-301& R-302). The last part is to purify isobutanol by azeotropic distillation(T-401,D-401＆ D-402). Furthermore, we wish we could apply our project to industry some other day. Hoping that the enormous production could be an alternative of gasoline.