Construction of an electrophilic E. coli strain

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.

E. coli KRX ΔdcuB::oprF strain

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.

Preparation of knockout deletion cassette

We create an E. coli knockout strain with GeneBridge. We decide to knock out C4 carboxylate transporter dcuB to prevent succinate export. Besides we insert outer membrane porines OprF (BBa_K1172507) into the genome. Both could be realized with Genebridge RedET-System. For simultaneous knockout and insertion a deletion cassette hast to be designed and established. We used overlap extension PCR to amplifie the complete deletion cassette (Bryksin & Matsumura, 2010). We used kanamycin as antibiotic selective marker amplified from the plasmid Flp705 using the Genebridge RedET-System protocoll. We designed Primer (BBa_K1465107, BBa_K1465108, BBa_K1465109, BBa_K1465110) with complementary 5´extensions for both fragments to connect them in overlap extension PCR. The amplified deletion cassette has homologous sites for recombination with the dcuB gene in E. coli KRX genome.

PCR analysis

Gene of C4 carboxylate transporter dcuB, which exchange fumarate against succinate, is about the size of 1341 bp. We amplified genome area of dcuB gene with primer, which bind about 530 bp upstream and 520 bp downstream of dcuB gene. So E. coli KRX bring out a PCR product of about 2391 bp, which could be demonstrated by agarose gelelectrophorese. However E. coli KRX knockout strain (E.coli KRX ΔdcuB::oprF) show a 4046 bp PCR product analyzed by agarose gelelectrophoresis. The size Figure XXX shows the result of agarose gelelectrophoresis with E. coli KRX wildtype and the modified strain E.coli KRX ΔdcuB::oprF. PCR product of E.coli KRX ΔdcuB::oprF genome amplified with primer dcuB_del_kon1 and dcuB_del_kon2 (4046 bp) is composed of antibiotic cassette (1637 bp), BBa_K1172507 (1359 bp) and upstream and downstream spacer elements (520 bp and 530 bp).

Sequencing

DNA sequencing of deletion cassette from E.coli KRX ΔdcuB::oprF shows expected results.

NPN-Assay

Activity test of outer membrane porin OprF (BBa_K1172507) in E.coli KRX ΔdcuB::oprF was investigated with NPN-Uptake-Assay (Cheng et al., 2005).
1-N-Phenylnaphthylamine (NPN) changes fluorescence activity between aqueous and hydrophobic milieu. There is only minor fluorescence in aqueous solution, but the transport in hydrophobic periplasmatic space causes an increased fluorescence. So NPN fluorescence is a good indicator for membrane permeability. Expression of outer membrane porines OprF effect an increasing membrane permeability.(Loh et al., 1984)

Figure XXX: Results of NPN-Uptake-Assay. Comparison between Escherichia coli KRX Wildtype and Escherichia coli KRX with BBa_K1172507, BBa_K1172502 and Escherichia coli KRX with genome integrated oprF gene (E.coli KRX ΔdcuB::oprF).

Figure XXX shows the result of NPN-Uptake-Assay. Higher fluorescence emission and higher membrane permeability could be observed with increasing promotor strength for OprF. Genome integrated oprF shows mid-level fluorescence emission and membramne permeability. Highest fluorescence level could be measured with oprF gene on high-copy pSB1C3 plasmid under control of T7 promotor.
Genome coded OprF show almost the same membrane permeability as constitutive expressed oprF with BBa_K1172507. Even there seems to be a marginal higher expression of genome integrated oprF in contrast to plasmid coded oprF although se same constitutive promotor (BBa_J23104) is used. This could be explained by physiological condition of the cell. Constitutive expression of high-copy oprF causes cell stress. Protein expression could be automatically downregulated by the cells. Single-copy genome integrated oprF show minor expression level, therefor cell stress is also smaller. As a consequence E. coli cells show a more effective expression of outer membrane porin OprF adjusted on physiological cell condition.

Phenotypic characterization with Biolog® system

Biolog® Microbial ID System is a simple and fast method for characterization of different bactaria, yeast and fungi species. It based on respiration in the presence of different metabolic relevant substances for expample carbon sources, nitrogen sources or toxines. The system uses redox chemistry wherein a tetrazolium dye become reduced when significant respiration occurs. Tetrazolium dye change its colour, because the cells generate reductive conditions in the course of electron transport chain. Tetrazolium cation become reduced to formazan by dehydrogenase from respiratory complex I. This allow a statement if respiration occurs in the presence of different substances.

E. coli KRX Wildtype and E.coli KRX ΔdcuB::oprF were incubated in Biolog® system to test respiration in the presence of fumarate. It has to be shown that E.coli KRX ΔdcuB::oprF shows no activity in the presence of fumarate because knockout of C4 carboxylate transporter dcuB makes fumarate uptake impossible for E. coli cells.

The results of Biolog® analysis are shown in figure XXX.

Fumarate reductase

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

Biolog® Microbial ID System is a simple and fast method for characterization of different bactaria, yeast and fungi species. It based on respiration in the presence of different metabolic relevant substances for expample carbon sources, nitrogen sources or toxines. The system uses redox chemistry wherein a tetrazolium dye become reduced when significant respiration occurs. Tetrazolium dye change its colour, because the cells generate reductive conditions in the course of electron transport chain. Tetrazolium cation become reduced to formazan by dehydrogenase from respiratory complex I. This allow a statement if respiration occurs in the presence of different substances.

We tested the influence of fumarate on Escherichia coli KRX wildtype and E. coli KRX with BBa_1465102. To show activity of fumarate reductase under controll of T7 promotor (BBa_1465102), we incubated cells with fumarate in Biolog® system for 48h. Results of Biolog ® analysis are shown in figure XXX.

E. coli KRX wildtype and E. coli KRX with BBa_1465102 show respiratory activity in the presence of fumarate. As unexpected E. coli KRX wildtype shows higher activity on fumarate as compared to BBa_1465102. Respiration of E. coli KRX with BBa_1465102 seems to be fewer and occurs subsequently in comparison to wildtype.

Neutral Red

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.

Bromphenol Blue

Cytochroms

Reference

• Iverson et al., 1999. Structure of the Escherichia coli Fumarate Reductase Respiratory Complex. Science, vol. 284, pp. 1961-1966
• Cheng et al., 2005. New antibiotic peptides, useful in treating or preventing a microbial or viral infections or in inactivating Gram-positive and -negative bacteria, protozoa, fungi and virus. patent DE10360435 (A1) ― 2005-07-28
• Loh et al., 1984. Use of the fluorescent-probe 1-Nphenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer-membrane of Pseudomonas aeruginosa. Antimicrob. Agents Chemother., vol. 26, pp. 546-551