Team:NCTU Formosa/BP discussion

In our experiment, we culture E.coli expressing foreign genes in a media with glucose, then did test by using two kinds of competent cell, DH5α and EPI300, and one circuit that contain kivd . Comparing these two different host cells, we find an obvious outcome of isobutanol production. DH5α is more efficient than EPI300, so we chose DH5α as our host cell.

To achieve high productivity of the target foreign products, it is desirable to seek pathways that are compatible to the host.
To produce isobutanol, the alss, ilvC, ilvD genes under the control of the Plac promoter on a plasmid were overexpressed to enhance 2-ketoisovalerate biosynthesis. The amplified ilv pathway was then combined with the producing pathway (Kivd ) to achieve isobutanol production.
Then we control the temperature in E.coli culture, as what we expected that the production of isobutanol will increase with lower incubation temperature.

Concluding above, we make the gene expression under temperature control. High temperature contribute to low production of cytotoxic isobutanol but high accumulation of the non-toxic intermediate. When we control the temperature to lower degree, the production of isobutanol will increase. In this way, we successfully gain our target product with the most efficient method.

Following is about the strains where the alss, ilvC, ilvD and Kivd genes are cloned from, with their point mutation sites: