Team:TU Delft-Leiden/Project/Life science/EET/characterisation

Module Electron Transport – Characterization

Our BioBrick BBa_K1316012 encodes the mtrCAB genes under control of an adjusted T7 lac promoter. The MtrC, MtrA and MtrB proteins form a conduit to transfer electrons across the membrane. To complete the implementation of the extracellular electron pathway we constructed BBa_K1316011 as well , a BioBrick encoding the ccm genes under control of the pFAB640 promoter. The ccm proteins help to mature the MtrCAB conduit. The elaborated combination of promoters and coding sequences that we used was found to generate the largest maximal current [1]. To test if this is true, we characterized BBa_K1316012 and BBa_K1316011 . We made use of SDS-page and UV-vis to see if the proteins are expressed. In addition, we made use of a potentiostat to do advanced current measurements. In this way we were able to visualize expression of ccm genes, and showed that induction of our BioBricks indeed results in a current flow.

Characterization of the Ccm genes

The CCM cluster is a cluster consisting of genes encoding for several (parts of) proteins. The cytochrome C maturation (Ccm) system consists of heme delivery proteins that help the conduit proteins, such as the proteins in the MtrCAB operon, to mature properly by translocating heme in the periplasm and catalyzes the formation of thioether bonds that link heme to two cysteine residues. The axial ligands are then coordinated to the heme iron and the holocytochrom C is folded. In these strains, no conduit proteins are inserted, but the heme delivery proteins should be more highly expressed. When there is an increased expression of heme delivery proteins, that can already be seen by the redness of the pellets (because of the heme) after the membrane purification.

Membrane purification and UV-VIS

To look into the expression of the cytochrome c maturation (Ccm) proteins, an UV-VIS spectrum has been recorded. E. coli (C43) cultures were transformed with BBa_K1316011 and then cultivated aerobically. E. coli (C43) Ccm+MtrCAB and E. coli (C43) without plasmid were used as a positive and negative control, respectively. E. coli (C43) Ccm+MtrCAB already has the Ccm system and the MtrCAB operon, so is expected to have expression of MtrA, MtrB and MtrC proteins because of the Ccm system. BBa_K1316011 is expected to have more expression of the Ccm proteins than the normal E. coli (C43) strain and more equal expressions of Ccm proteins compared with E. coli (C43) Ccm+MtrCAB because of the inserted Ccm operon. When there is an increased expression of heme delivery proteins, that can already be seen by the redness of the pellets (because of the heme) after induction with IPTG.

Before membrane protein purification, BBa_K1316011 and the controls where induced with IPTG. Visual analysis of the pellets shown that there was a difference in color in BBa_K1316011 and E. coli (C43) Ccm+MtrCAB compared with E. coli (C43). The BBa_K1316011 and E. coli (C43) Ccm+MtrCAB had a red color, which could be indicating the increased production of cytochrome c proteins because of the heme delivery proteins. Membrane protein purifications where done by low-speed and high-speed centrifugation.

According to [2], there should be a peak around 550nm for cytochrome c proteins. Using the UV-VIS results, there is a peak for all membrane fractions around 550nm, so it is possible to confirm the expression of cytochrome c proteins in all the samples. There is a difference between E. coli (C43) without plasmid and BBa_K1316011, as shown in figure 1. These observations confirm that BBa_K1316011 and E. coli (C43) Ccm+MtrCAB offer enhanced cytochrome c expression compared to the E. coli (C43) strain without the Ccm or MtrCAB plasmids.

SDS-PAGE

An SDS-PAGE has been done with the membrane fractions for E. coli (C43), E. coli (C43) Ccm+MtrCAB and BBa_K1316011. As mentioned before, no conduit proteins, such as the proteins in the MtrCAB operon, are included in BBa_K1316011, but they are included in E. coli (C43) Ccm+MtrCAB. When the MtrCAB operon is included, the membrane fractions of E. coli (C43) Ccm+MtrCAB should contain MtrA, a 32-kD periplasmic decaheme cytochrome c, MtrC is a 69-kD cell-surface-exposed lipid-anchored decaheme cytochrome c and MtrB is a 72-kD predicted twenty-eight strand β-barrel outer membrane protein.[1]

There are no significant differences between BBa_K1316011 and both positive and negative controls observed using SDS-PAGE. There are no MtrCAB proteins observed when looking into the SDS-PAGE lane for E. coli (C43) Ccm+MtrCAB. Due to the the weak MtrCAB promotor, the expression of MtrCAB proteins might be too low to be visible on the gel.

Bioreactor

Shewanella oneidis MR-1 uses the MtrCAB proteins, the principal proteins in this module, to extracellularly reduce bulky metal oxide crystals which it uses as terminal electron acceptors in its respiration. Electrons stem from the intracellular oxidation of (organic) electron donors, and the process is thermodynamically favourable under physiological conditions. In this project we don’t seek to reduce metal-oxides but rather a working electrode in a three electrode cell.

Introduction to voltammetry

The three electrode cell is used to perform voltammetry which is an electro analytical method used to investigate the half-cell reactivity of an analyte. In voltammetry potential-difference (E) between a working and a reference electrode in an electrochemical cell is controlled and the resulting current (I) is measured. The working electrode is in physical contact with the analyte thereby facilitating the transfer of charge when a potential is applied. The reference electrode has a known, stable electrode potential and is used to gauge the potential of the working electrode. The third electrode is the auxiliary (or counter) electrode which balances the charge in the cell; it reduces or oxidizes any molecules that are in the solution. When no red-ox reactions take place at the working electrode, only a marginal current flows because of the applied potential between the reference and working electrode due to electrostatic effects. When the working electrode is either reduced or oxidized electrons flow through the circuit which can easily be detected using an Amperometer. In most voltammetric experiments the potential is varied at differing rates over time, however in this set-up the potential is kept constant for the course of the experiment. When a positive potential is applied to the working electrode in our set-up, electrons present on the extracellular side of the outer membrane of our engineered E.coli reduce it hence: a current flows. More on the potentiostat that our team can be found in the gadget subsection [LINK].

Our bioreactor

Figure x shows a schematic representation of our bioreactor. The working electrode is made of a square piece of carbon cloth [REF] which is folded and tied together with a tie wrap to make it fit in the bioreactor. Carbon cloth has a large surface to volume ration, is non-toxic and therefore ideal for voltammetry handling live organisms. The counter electrode is made of a graphite rod that is wrapped in silicon tubing to prevent any shorts due to the two electrodes touching. The reference electrode is silver/silver chloride (Ag/AgCl) with a saturated KCl electrolyte solution, yielding an electrode potential of Eref= +0.197 V versus a Standar Hydrogen Electrode (SHE)[REF]. When a working electrode potential of for instance E = 0.2V is applied this means that the potential of the working electrode is actually E = 0.2 + 0.197 = 0.397V vs SHE. The temperature in the bioreactor is controlled through a heat mantle around the compartment where the cells are situated which is fed with warm water from a warm water-bath. The broth in the bioreactor is stirred with a magnetic stirrer, and there is a sampling tube present to take samples for OD600 measurements. Due to the nature of the cascade of reactions yielding the electrons that finally reduce the working electrode the broth needs to be completely anoxic, as pointed out by the modelling of the carbon metabolism [LINK]. To keep the broth free of oxygen a gas inlet is attached to a needle which feeds sterile N2 into the reactor close to the stirrer. To depressurize the reactor also a gas outlet is present. A picture of our bioreactor to which all above-mentioned components attached, and pictures of the individual components is shown in figure x.

Metabolism and the source of electrons for the MtrCAB pathway

There is but a limited scope of substrates that can act as electron donors for the MtrCAB pathway which are: lactate, N- Acetylglucosamine, formate, and hydrogen. In our experiments lactate is used as an electron donor since, when present at relatively high concentrations, it is dehydrogenated by lactate-dehydrogenase (LDH) to pyruvate and yielding NADH as seen in reaction:

The NADH is then oxidized to produce menaquinol which then yields its electrons to the MtrCAB proteins via E. coli’s native NapC. Other carbon substrates like glucose ferment for which reason these substrates do not yield an excess of NADH which is essential for fuelling the MtrCAB pathway. To prove this principle we also used glycerol as a carbon source instead of lactate which can be fermented anaerobically, therefore theoretically yielding no electrons for the MtrCAB pathway. For more information on the carbon metabolism see [LINK].

The experiments

In the first experiment we tried to roughly replicate the conditions as stated in the Jensen [REF] article; the exact protocol for seeding the bioreactor can be found in the protocol for bioreactor [LINK]. Figure x represents the current in mA divided by the first OD600 measurement at the start of the experiment; figure x represents the OD600 measurements over time, for which the raw data can be found here . OD600 is not directly correlated to current, so only the first OD600 measurements is used to normalize the data for comparison. Cells in all experiments are grown in M4 minimal medium supplemented with 40mM D/L-lactate, except for one measurement where the cells were grown in M4 with 40mM of glycerol. E.coli C43 bearing the I5023 and the C640 plasmids is referred to as the Ajo-F strain, and 'Empty' cells are untransformed E.coli C43 which serve as a negative control. The I5023 plasmid contains the heme-loading Ccm proteins while the C640 plasmid encodes the MtrCAB proteins.

Figure xA shows a significant difference in current measured between the empty C43 strain and the Ajo-F strain that was induced overnight. The difference is roughly 0.7mA at the beginning of the experiment, but this difference decreases over time. This decrease might be due to the faster drop in OD600 of the Ajo-F strain compared to the C43 strain. If faster decrease of OD600 were to be the explanation for decreasing current, it proves that the 'concentration' of cells is correlated to the observed current. Since the only difference in experimental procedure is the presence of the Ccm and MtrCAB proteins in the Ajo-F strain this result suggests that the observed current is indeed due to the functioning of these proteins. When the Ajo-F strain was resuspended in M4 medium supplemented with 40mM glycerol it showed the exact same current as the empty C43 strain, proving that current was observed because of above-mentioned lactate dehydrogenation and subsequent steps leading to the excretion of electrons.

Figure 4B shows that there is an even more significant difference in observed current between 'empty' C43 cells and the Ajo-F strain at working electrode potential E = 0.2V. This is of interrest because the aim is to make a biosensor that can produce quantative data, and therefore a larger contrast is more preferable. Once again a steep drop in OD600 values can be detected in the Ajo-F strain that was induced overnight as shown in figure x. This drop in OD600 is again roughly correlated to the decrease in current, proving that current is produced by live cells. The OD600 of 'Empty' C43 cells drops at a more gentle slope, indicating that cells are dying at a slower rate. The difference in the rate of decline in OD600 values between C43 cells with and without MtrCAB might be due to one of two reasons; the first reason being that MtrCAB proteins make the membrane more susceptible to tears caused by electrostatic effects. Chargen species inside the cell

References

1. C.P. Goldbeck et al., Tuning promoter strengths for improved synthesis and function of electron conduits in E. coli, ACS Synth. Biol. 2 (3), pp 150–159 (2013)

2. Reedy, C.J. & Gibney, B.R. et al., Heme protein assemblies, Chem Rev 104 (2), pp 617–49 (2004)

Frederik Golitsch, Clemens Bücking, Johannes Gescher, Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexes. Biosensors and Bioelectronics, 47, pp 285–291 (2013)