Team:NCTU Formosa/Project

Overall project

Our team invented an enzyme pathway for E. coli to produce butanol, which is closely related chemically to ethanol. However, the two extra carbon atoms and their attendant hydrogens let it be a higher energy value. In terms of storage and handling, it is less corrosive than ethanol and has several other advantages. Therefore, it may be a better substitution to ethanol as a biofuel.

The pathway starts at glucose, through glycolysis it will convert to pyruvate. Then the pyruvate goes through enzymatic process and produces isobutyraldehyde, which is toxic to the E.coli. To prevent the toxicity harming E.coli, we use the temperature regulation system. The first part is The 37℃ part. Our team makes E. coli produce a lot of nontoxic 2-Ketoisovalerate. Afterwards we reduce the temperature to 30℃.The 30℃ part converts 2-ketoisovalerate to isobutyraldehyde at one blast. Then through simple process we get isobutanol, the potential biofuel in the future.

What worth mentioning is that our team adds various zinc fingers before each enzyme to increase the affinity and efficiency of gene expression. Also, we are planning to design a container to produce and concentrate isobutanol. NCTU_Formosa hopes to soon provide an affordable and “green” transportation fuel to the world!

Project Details

The process of our experiment can be summarized by the following process.



Because one of the products of our experiment is harmful to the E.coli ,we want to resolve the problem. If the problem did’t disappear ,we would let E.coli be degraded. Then we decided to use 37℃ system to regulate our E.coli.

First ,we make E.coli stay in 37℃ environment. E.coli will produce tetR to inhibit the production of isobutyraldehyde which is toxic to E.coli.

After all of 2-ketoisovalerate are produced completely , we begin to let E.coli stay in 30℃ environment. The change will make E.coli begin the next reaction. It can be showed by the following process.

Finally, we can obtain the main product, isobutanol, from the reaction.Our resolution is to promote all 2-ketoisovalerate turn into the toxic intermediate at a time for decreasing the strength of E.coli inhibition from isobutyaldehyde.

Result

ZINC FINGER

A zinc finger is a kind of finger-shaped protein that contains one or more zinc ions to stabilize the structure. Proteins that contain zinc fingers(zinc finger protein) are the most common transcription factors in organisms. A transcription factor is a protein that binds to DNA and controls the expression of genes. The zinc finger proteins bind specific DNA, RNA, or other molecules with the interaction of its domain and these molecules.

We add zif268,PBSII,HIVC and Gli1 four zinc fingers separately in front of our biopart: Alss, ilvc, ilvD, kivd. We expect that the enzyme with zinc fingers would increase the productivity of isobutanol.

zif268

Zif268 Binding With DNA

This zinc finger identifies the DNA sequence 5'-GCG TGG GCG-3',binding to the certain domain of the sequence.

PBSII

Identifies DNA sequence 5'-GTG TGG AAA-3'.

HivC

Identifies DNA sequence 5'-GAT GCT GCA-3'.

Gli1

Five-Finger GLI/DNA Complex

Identifies DNA sequence 5'-GAC CAC CCA-3'.