Usage and Biology
Isobutanol is an important substance for industry. No known organism can produce isobutanol or other branched-chain alcohols. Atsumi et al. presented a metabolic pathway to produce isobutanol in Escherichia coli. The pathway is shown in Figure 1.
The steps in the conversion of pyruvate to 2-ketoisovalerate can be executed by proteins existing in E. coli (IlvIH, IlvC and IlvD). Since E. coli also has an alcohol dehydrogenase (AdhE), the only required protein for the isobutanol production is a ketoisovalerate decarboxylase. This protein (KivD) can be received from Lactococcus lactis. The pathway shown in Figure 1 is already an improvement of the described way. The native protein IlvIH is replaced by the AlsS from Bacillus subtilis to increase the isobutanol production. (Atsumi et al.)
As we want to integrate this pathway in E.coli we used and improved existing BioBricks from the iGEM team NCTU Formosa 2011/2012. We used gene coding sequences of four out of five required proteins for the isobutanol production.
These genes are
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 5669
Illegal XhoI site found at 4949
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 2332
- 1000Illegal BsaI site found at 1918
Illegal BsaI site found at 4735
Illegal BsaI site found at 4992
Illegal BsaI.rc site found at 320
Illegal BsaI.rc site found at 914
Illegal BsaI.rc site found at 2737
For characterization we analyzed the protein expression of BBa_K1465302 under the control of T7 promoter we made a cultivation of E. coli KRX with the constructs.
Samples of E. coli KRX with our construct pSB1A2_T7_alsS_ilvC_ilvD_kivD were taken. Protein expression was induced with rhamnose when the culture reached a OD600 of 0.8. The first sample was taken right before the induction. Additionally we took samples two, four, 21 and 23 hours later. With these samples, we made a SDS Page. Figure 1 shows the picture of this SDS Page.
These observations fit to our expectations of possible results of this experiment, because all proteins seem to be overexpressed.