Primer

Part:BBa_K1465110

Designed by: iGEM-Team Bielefeld 2014   Group: iGEM14_Bielefeld-CeBiTec   (2014-10-05)

dcuB rev

Primer dcuB rev (BBa_K1465110) is part of the construction of an electrophilic E. coli strain, which metabolism could be increased by growing under influence of electric power. It is part of a primer collection containing BBa_K1465107, BBa_K1465108, BBa_K1465109 and BBa_K1465110. The aim is to create a deletion cassette for knocking out C4 carboxylate transporter DcuB and insert outer membrane porine OprF (BBa_K1172507) into E. coli chromosome in the same step.

Usage and Biology

C4 Carboxylate Antiporter DcuB

Under anaerobic conditions E. coli cells use different alternative electron acceptors instead of oxygen. Partially the bacteria use fumarate respiration, whereby fumarate is reduced into succinate. There are also other potential less-oxidizing substances for bacteria to release their electrons, for example anorganic compounds like nitrate (NO3-) or sulfate (SO42-).(Gottschalk et al., 1986) Fumarate respiration leads to succinate excretion through the C4 carboxylate transporter DcuB. It is an antiporter which exchanges fumarate against succinate under anaerobic conditions. Under aerobic condition there is usually no succinate release observed. In connection to the carbon dioxide fixation in our second module we planned on working under oxygen limiting conditions, hence effective carbon dioxid fixation is possible. So in case of oxygen limiting conditions, there could occured partial fumarate respiration in E. coli. Besides there was shown activity of DcuB antiporter in the presence of high fumarate concentrations (Janausch, 2001). To achieve an effective electron uptake and prevent any succinate excretion, the C4 carboxylate antiporter DcuB has to be knocked out in our E. coli strain.
We planned a targeted knockout of the dcuB gene in E. coli KRX using the Genebridge Red/ET-System. In the same step we are going to integrate the outer membrane porine OprF (BBa_K1172507) into the bacterial chromosome under controll of a constitutive promotor (BBa_J23104). This ensure the permeability of the outer membrane and avoid a plasmid overload of the bacteria, because for our system the outer membrane porines are indispensable.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

Construction of an electrophilic E. coli strain

We construct an E. coli strain, which is able to accept electrons stimulating its metabolism. Direct electron transfer in bacteria is a very complex and not completely cleared up so far. Due to this we focused on the indirect electron transfer via a mediator, which is reduced at the electrode in a electrobiochemical reactor and would be reoxidized again by bacterial cells.(Park et al., 1999) In gram-negative bacteria E. coli there are two membranes with a periplasmatic space between them, which has to be overcome in the course of the electron transfer.
We worked on three different mediators: neutral red, bromphenol blue and cytochromes. Additionally we constructed an electropholic E. coli strain, which shows an increased metabolic activity growing with electric power, which has been proven in our H-cell reactor (Park et al., 1999).
The electron transfer system consists of different steps. First of all the reduced mediator has to cross the outer membrane of the E. coli cell. For that we used the outer membrane porine OprF (BBa_K1172507) provided by the iGEM Team Bielefeld-Germany 2013. Crossing the periplasmatic space, the mediator adsorb at the inner membrane of the E. coli cell. The mediator functions as an electron donor for the 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 the succiante dehydrogenase. Succinate excretion is avoid, because of the knockout of the C4 carboxylate transporter DcuB. In the reaction of succinate dehydrogenase electrons are transferred to FAD+ generating FADH2, which enter the electron transport chain. The electron transport facilitates proton translocation over the inner bacterial membrane. The proton motoric force is used by ATP synthase. Generated ATP and reductive power in the bacterial cell leads to an increasing metabolic activity.

E. coli KRX ΔdcuB::oprF strain

We investigated the effect of the C4 carboxylate transporter DcuB knockout on E. coli KRX. Furthermore we showed the integration of the outer membrane porine OprF (BBa_K1172507) into the bacterial genome by replacing the gene of E. coli C4 carboxylate antiporter DcuB. So we did knockout and insertion in a one-step process. The successful knockout of the DcuB antiporter and simultaneous insertion of BBa_K1172507 was shown with PCR analysis, DNA sequencing and phenotypic investigation via Biolog analysis and 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 decide to knock out the C4 carboxylate transporter DcuB to prevent succinate export. Besides we inserted the outer membrane porines OprF (BBa_K1172507) into the genome. Both could be realized with the Genebridge RedET-System. For simultaneous knockout and insertion a deletion cassette had to be designed and established. We used overlap extension PCR to amplify the complete deletion cassette (Bryksin & Matsumura, 2010). We used kanamycin as an antibiotic selective marker amplified from the plasmid Flp705 using the Genebridge RedET-System protocoll. We designed the primers (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 the E. coli KRX genome.

Verification of knockout and deletion cassette


Figure 1: Results of the colony PCR for
analysis of dcuB genome area
analyzed via agarose gelelectrophoresis.
E. coli KRX wildtype band
is shown on the rigth of about 2391 bp.
E.coli KRX ΔdcuB::oprF band is shown
on the left at 4046 bp.

The gene of the C4 carboxylate transporter DcuB, which exchange fumarate against succinate, has a size of 1341 bp. We amplified the genome area of the dcuB gene with primers (dcuB_del_kon1 and dcuB_del_kon2), which bind 530 bp upstream and 520 bp downstream of the dcuB gene. When using the E. coli KRX wildtype as a template the resulting PCR product has a size of 2391 bp, which could be demonstrated by agarose gelelectrophoresis. However the E. coli KRX knockout strain (E.coli KRX ΔdcuB::oprF) showed a 4046 bp PCR product analyzed by agarose gelelectrophoresis. Figure 1 shows the result of the agarose gelelectrophoresis with E. coli KRX wildtype and the modified strain E.coli KRX ΔdcuB::oprF.

The PCR product of E.coli KRX ΔdcuB::oprF genome amplified with primer dcuB_del_kon1 and dcuB_del_kon2 (4046 bp) and is composed of the antibiotic cassette (1637 bp), BBa_K1172507 (1359 bp) and upstream and downstream spacer elements (520 bp and 530 bp).

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









NPN-Assay

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



Figure 2: Results of the 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 2 shows the results of the NPN-Uptake-Assay. Higher fluorescence emission and higher membrane permeability could be observed with increasing promotor strength for oprF. The genome integrated oprF shows mid-level fluorescence emission and membrane permeability. Highest fluorescence levels could be measured with the oprF gene on a high-copy pSB1C3 plasmid under control of the T7 promotor.
Genome integrated oprF showed almost the same membrane permeability as the constitutive expressed oprF with BBa_K1172507. There even seems to be a marginal higher expression of the genome integrated oprF in contrast to the plasmid coded oprF although the same constitutive promotor (BBa_J23104) is used. This could be explained by physiological condition of the cell. Constitutive expression of the high-copy oprF causes cell stress. Protein expression could be automatically downregulated by the cells. The single-copy genome integrated oprF showed a reduced expression level, therefor cell stress is also smaller. As a consequence E. coli cells showed a more effective expression of the 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 the characterization of different bacteria, yeast and fungi species. It is based on respiration in the presence of different metabolic relevant substances for example carbon sources, nitrogen sources or toxines. The system uses redox chemistry wherein a tetrazolium dye is reduced when significant respiration occurs. Tetrazolium dye change its colour, because the cells generate reductive conditions in the course of the electron transport chain. A tetrazolium cation is reduced to formazan by dehydrogenase from the respiratory complex I. This allow a statement if respiration occurs in the presence of different substances.

The E. coli KRX wildtype and E.coli KRX ΔdcuB::oprF were incubated in the Biolog® system to test respiration in the presence of fumarate. It was expected that the E.coli KRX ΔdcuB::oprF shows no activity in the presence of fumarate because of the knockout of the C4 carboxylate transporter dcuB, which makes fumarate uptake impossible for E. coli cells.
The results of the Biolog® analysis are shown in figure 3.


Figure 3: Results of the Biolog® analysis of E.coli KRX ΔdcuB::oprF in comparison to Escherichia coli KRX wildtype. Respiratory activity is shown under influence of fumarate.

Biolog® analysis showed that there is no significant respiratory activity of E.coli KRX ΔdcuB::oprF in the presence of fumarate during the first 35 hours of incubation. Later on respiration occurs up to 50% of maximal respiratory activity of the E. coli KRX wildtype after 48 hours of incubation at 37°C.
The results in figure 3 demonstrate that the knockout of C4 carboxylate antiporter dcuB was successful. Respiration activity after 35 hours could be explained by a change in metabolism of E. coli cells. Based on cell stress in minimal medium bacteria use other ways for uptake of fumarate. It is possible that another transporter compensates the activity of the DcuB transporter, but activation and expression of alternative pathways require a space of time, espacially under limited conditions in minimal medium. This could explain the delayed respiration of E.coli KRX ΔdcuB::oprF.

Anaerobic cultivation

We cultivated E. coli KRX ΔdcuB::oprF under anaerobic conditions. We used M9 minimal medium with 50 mM glucose. Among growth characteristic we focused on metabolite analysis. Under anaerobic conditions E. coli cells use different alternative electron acceptors instead of oxygen. In part the bacteria use fumarate respiration, whereby fumarate is reduced into succinate. There are also other potential pathways for bacteria to release their electrons, for example anorganic compounds like nitrate (NO3-) or sulfate (SO42-).(Gottschalk et al., 1986) We analyzed succinate concentration and glucose concentration in the culture supernatant via HPLC.

We expect succinate production of the E. coli KRX wildtype, but E. coli KRX ΔdcuB::oprF should not release succinate into the media because the C4 carboxylate transporter DcuB is knocked out. As a positive control we use E. coli ΔdcuB749::kan, an E. coli strain with a reviewed knockout in dcuB gene. This strain also should not show any succinate transport into the medium.



Figure 4: Results of the anaerobic cultivation of Escherichia coli KRX wildtype in M9 minimal medium with 50 mM glucose. Glucose and succinate concentration were measured in duplicates with HPLC.


Figure 5: Results of the anaerobic cultivation of Escherichia coli ΔdcuB749::kan in M9 minimal medium with 50 mM glucose. Glucose and succinate concentration were measured in duplicates with HPLC.


Figure 6: Results of the anaerobic cultivation of Escherichia coli KRX ΔdcuB::oprF in M9 minimal medium with 50 mM glucose. Glucose and succinate concentration were measured in duplicates with HPLC.


Figure 7: Results of the anaerobic cultivation of Escherichia coli KRX ΔdcuB::oprF in M9 minimal medium with 50 mM glucose as compared to Escherichia coli KRX wildtype. Glucose and succinate concentration were measured in duplicates with HPLC.

Figure 4 shows the production of succinate by the Escherichia coli KRX wildtype under anaerobic condition as expected. Succinate is released into the medium via the C4 carboxylate transporter DcuB. Our constructed E. coli KRX ΔdcuB::oprF strain shows no succinate export under anaerobic conditions displayed in figure 6. This demonstrates a successful knockout of the dcuB gene in the E. coli KRX ΔdcuB::oprF strain. As positive control we got an E. coli strain from the KEIO collection with a verified knockout in the dcuB gene.
Figure 5 shows the result of the anaerobic cultivation and succinate concentration measurement of this E. coli strain. A very low succinate concetration in the culture supernatant could be observed. In comparison to the E. coli KRX wildtype there is a very low succinate export. Only traces of succinate could be measured attributed to transport of succinate by other transporters with reduced specifity.
Furthermore in Escherichia coli ΔdcuB749::kan the knockout is not over the complete dcuB gene. Insertion of kanamycin selection cassette was executed at position 749 of dcuB gene. So there could be a residual activity of DcuB because of residual expressed parts of the antiporter protein. However the knockout of the dcuB in E. coli KRX ΔdcuB::oprF is complete and residual activity is not possible as shown in figure 7. Comparison between the E. coli wildtype and E. coli KRX ΔdcuB::oprF is an obvious demonstration of a completely functional knockout of the C4 carboxylate antiporter DcuB.

Methods

NPN membrane permeability assay
  • inoculate 20 ml of an overnight culture with 1 ml preculture
  • incubate cells at 37 °C until OD600 of 0.5
  • centrifugation of whole culture volume (10 min at 3000 g)
  • discard entire supernatant
  • resuspend pellet in the same volume PBS Buffer
  • repeat washing steps 3 to 5 twice
  • mix washed cells with 10 mM NPN stock solution to a final concentration of 10 μM in a UV cuvette
  • fluorescence measurement with Shimadzu fluorescence spectrophotometer
    • sample volume: 1 ml
    • Excitation: 355 nm
    • Emission Bandwidth: 380 - 500 nm

    Anaerobic cultivation

    • Whole anaerobic work takes place in a two-hand Glove Bag.
    • All media and buffer has to be degased with nitrogen (N2) gas via a sparger befor starting cultivation.
    • Grow preculture under aerobic condition at 37°C.
    • Cells were cultivated in a gas-tight 15 ml tube additional sealed with parafilm.
    • Cultivation volume is about 10 mL. Fill cultivation tube with 8.5 ml steril degased cultivation medium.
    • When preculture reaches OD600 of 0.6-0.8, take 1.5 ml into a steril tube.
    • Centrifugate 1 min at 5,000 rpm and discard complete supernatant.
    • resuspend pellet in 1.5 ml steril degased PBS buffer.
    • Repeat previous step but resuspend in steril, degased cultivation medium. This two steps avoid residual oxygen in the inoculum.
    • Inoculate culture with 1.5 ml washed and resuspended cells at OD600 of 0.1.
    • Tubes are filled and opened for sampling only under nitrogen atmosphere.
    • Tubes are incubated in a shaker at 37°C.
    • At the beginning of anaerobic cultivation residual oxygen from preculture would be consumed. Therefore faster growth could be observed in the first period of cultivation until cells shift to anaerobic metabolism.
    • There should be more frequent sampling at the beginning about 4 to 6-hour intervalls (two times) after inoculation.
    • Regular sampling takes place at 1-day intervalls.
    • Sampling volume is about 1 mL, using 500 μl for OD600 measurement and 500 μl for HPLC analysis.
    • After sampling process tubes are overflowed with nitrogen gas, tightly closed and sealed with parafilm again.
    • Tubes are further incubated in a shaker at 37°C.

    Biolog System

    This protocol is based on the technical guide from Biolog

    • Grow the strain of interest on a BUG+B (Biolog Universal Growth Medium + 5 % Sheep blood) agar plate by streaking out for single colonies
    • Incubate overnight at 37 °C
    • Pick single colonies with sterile swab
    • Transfer them into sterile tube containing 10 ml 1.0x IF-0a (mix 8 ml 1.2x IF-0a with 2 ml sterile water) to adjust transmittance to 42 %. This creates solution A.
    • Mix 144 μl Dye Mix A for Gram-negative bacteria and 1.856 ml sterile water with 8 ml 1.2x IF-0a solution. This creates solution B.
    • Mix 1.8 ml of solution A with 9 ml of solution B to achieve a final cell density of 85 % transmittance. This creates solution C.
    • Inoculate Biolog PM plate (96 wells) with 100 μl suspension C per well incubate loaded plate for 48 h in the OmniLog PM system

    References

  • Janausch, 2001. Rekonstitution des Fumaratsensors DcuS in Liposomen und Transport von Fumarat und Succinat in Escherichia coli. Doctoral dissertation at Johannes Gutenberg-Universität Mainz, Germany
  • 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
  • Gottschalk et al., 1986. Bacterial Metabolism. Springer-Verlag, New York, Berlin, Heidelberg, Tokyo
  • Park et al., 1999. Utilization of Electrically Reduced Neutral Red byActinobacillus succinogenes: Physiological Function of Neutral Red in Membrane-Driven Fumarate Reduction and Energy Conservation. Journal of Bacteriology, vol. 181, pp. 2403-2410