Short summary

Our first module deals with the construction of an E. coli strain, which is able to accept electrons stimulating its metabolism. We regard on two different electron transfer systems: direct and indirect electron transfer.
Direct electron transfer in bacteria is a very complex and not completely cleared up so far. So we focused on the indirect electron transfer via a mediator, which become reduced at the electrode in a electrobiochemical reactor and would be reoxidized again by bacterial cells. E. coli is a gram-negative bacteria and so there are two membranes with a periplasmatic space betwen them, which has to break through.
In module 1 we worked on three different mediators: neutral red, bromphenol blue and cytochromes. Additionally we construct an electropholic E. coli strain, which shows an increased metabolic activity growing with electric power as has been proved in our h-cell reactor. The electron transfer system consists of different steps. First of all the reduced mediator has to be cross the outer membrane of E. coli cell. For that we use outer membrane porine OprF (BBa_K1172507) provided by iGEM Team Bielefeld-Germany 2013. Crossing the periplasmatic space, the mediator adsorb at inner membrane of E. coli cell. The mediator functioned as electron donor for over expressed fumarate reductase. In this step succinate is produced in the cytoplasm. Reduction of fumarate into succinate creates a loop into the citric acid cycle, because succinate would be reoxidized again by succiante dehydrogenase. In that reaction electron are transferred to FAD⁺ generating FADH₂, which enter the electron transport chain. The electron transport achieve proton translocation over the inner bacterial membrane. The proton motoric force is used by ATP synthase. Generated ATP effects an increasing metabolic activity.

Short summary

We invastigated the effect of C4 carboxylate transporter dcuB knockout on E. coli KRX. Furthermore we show the integration of outer membrane porine OprF (BBa_K1172507) into bacterial genome by replacing gene of E. coli C4 carboxylate antiporter dcuB. So we did knockout and insertion in a one-step process. Successful knockout of dcuB antiporter and simultaneous insertion of BBa_K1172507 was shown with PCR analysis, DNA sequencing and phenotypic investigation, for expample Biolog analysis, anaerobic cultivation in M9 minimal media with fumarate supplemented. Substrates and products were analyzed by HPLC. The electrobiochemical behavior of E. coli KRX with knocked out C4 carboxylate antiporter dcuB was tested in a H-cell reactor.

PCR analysis

Sequencing

Phenotypic characterization with Biolog® system

Short summary

Upon the expression of fumarate reductase in E. coli we analyzed the metabolic behavior under aerobic and anaerobic conditions. Furthermore we characterize the impact on electrochemical behavior of E. coli. We show expression of fumarate reductase frd (BBa_1465102) using SDS-PAGE in combination with MALDI-TOF/MS. Activity could be shown with HPLC analysis of fumarate consumption and succinate production. Furthermore we investigated fumarate reductase activity in different E. coli strains by phenotypic MicroArray (PM) analysis with a Biolog® system.

SDS-PAGE

The fumarate reductase could be detected in purified membrane and periplasmatic protein fraction. Proteins were fractioned by cold osmotic shock of E. coli KRX at different steps after induction of protein expression. SDS-PAGE shows the expression of fumarate reductase in E. coli KRX under control of T7 promotor (BBa_1465102). Fumarate reductase consist of four subunits, two large catalytic and two smaller membrane associated subunits (Iverson et al., 1999). The two catalytic subunits could be detected via SDS-PAGE.

Anaerobic cultivation

We cultivate E. coli KRX under anaerobic conditions to characterize activity of fumarate reductase frd (BBa_1465102 under controll of T7 promotor. Fumarate and succinate concentrations were detected with HPLC.

Phenotypic characterization with Biolog® system

The Biolog system allows Phenotype MicroArrays (PM) to analyze cellular phenotypes all at once. It is possible to characterize the influence of a lot of different energy or carbon sources, trace elements, other supplements or toxins on bacterial cells. As well as gram positive and gram negative bacteria are able to use. We tested the influence of fumarate on Escherichia coli KRX wild type and different genetically modified E. coli cells. To show activity of over expressed fumarate reductase (BBa_1465102) under controll of T7 promotor, we investigated behavior of E. coli KRX wildtype, E. coli KRX with BBa_1465102 and KRX ΔdcuB::oprF with BBa_1465102 incubated with fumarate in Biolog system.

Short summary

We tested neutral red as a mediator for electron transfer into bacterial cells. The electrochemical behavior of neutral red was analyzed in a H-cell reactor.