Introduction to the project

Energy depletion of the earth has been a critical problem. We must fine 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

The report shows that we use fluorescence protein to mark the second part of our biobrick . We can see that fluorescence protein staying in 30°C environment will have higher expression than staying in 37°C environment . According to it , we can see that our system do truly work!

　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.

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

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

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

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　Instrument

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　Conclusion

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　Optimization

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　Future works

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