With our project we engage several problems. The first big problem is the lack of efficient storage and transport technology for electrical energy. The second problem is the increasing amount of carbon dioxide in the atmosphere. The third problem is the depletion of crude oil in the next decades.
Based on these problems we thought of a system which uses electricity and carbon dioxide to generate isobutanol. This system will be implemented in E. coli. For a better overview we divided our project in three modules.
In the first module we were working on a possibility to generate reduction equivalents by introducing a mediating substance which is reduced. The cell will oxidize the substance again which result in a cyclic system. We constructed two different fermenter to do different cultivations experiments. The first one is a traditional H-cell fermenter, the second one is a flow-cell. The H-cell fermenter was used intensively for cultivation experiments. We modified the metabolic pathway of fumarate by a knockout of the C4 carboxylate antiporter DcuB in E. coli and the overexpression of different fumarate reductases. Furthermore the outer membrane porine OprF was integrated into the bacterial chromosome to ensure a constitutive expression and reduce the plasmid overload of bacterial cells.
The second module aims to enable a functional Calvin cycle. In E. coli there are three enzymes missing to enable the whole cylce. The approach to transform the phosphoribulokinase A, the sedoheptulose 1,7-bisphosphatase and the RuBisCO was succesful and functionally tested. In addition we were able to transform a functional empty carboxysome which we verified through a GFP fusion. This could be used for further research as a biotechnological target because it is the only functional bacterial microcompartment in the parts registry up to our best knowledge. Concluding we were able verify the functionality of the RuBisCO in an in vitro assay. But we were not able to enable the whole cycle because of the lack of function of the prkA.
The third module tried to enable an isobutanol production pathway which was done before by the iGEM Team Formosa 2011 and 2012. We were able to transform all parts and improve the BioBrick by adding the alcohol dehydrogenase. We could verfiy the production of isobutanol via GC-MS.
In addition we implemented a antibiotic-free selection system.