Difference between revisions of "Part:BBa K1316012"

Revision as of 09:41, 17 October 2014

T7 lacO + mtrCAB

Combination of T7 promoter with the lac operator. For the promoter to be active both T7 RNA polymerase and the lac operon inducer (lactose or an analogue such as IPTG) must be present. This double regulatory system reduces the promoter leakage MtrCAB is an electron transport complex of the bacteria Shewanella oneidensis

Improvement of a part

We could not detect the coding sequence of mtrCAB by restriction analysis of Bielefeld 2013 iGEM team part BBa_K1172401 or by sequencing of Bielefeld 2013 iGEM team part BBa_K1172403 (mtrCAB with medium Promoter and medium RBS), which should contain the mtrCAB coding region of BBa_K1172401 according to Bielefeld 2013 iGEM team. Our team has created part BBa_K1316012 which contains the proper mtrCAB coding sequence on it and, therefore represents an improvement of BBa_K1172401 and BBa_K1172403 Bielefeld 2013 BioBricks.

Characterisation

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 BBa_K1316012 (MtrCAB + T7 pLac) insert in a non-biobrick backbone and BBa_K1316011 (ccm cluster + pFAB640) in a non-biobrick backbone is referret to as the Ajo-F strain, and 'Empty' cells are non-transformed E.coli C43 which serve as a negative control. The bio-bricks were assayed in non-bio-brick backbones because these have a low copy-number, which is important to not express the

Figure x 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 re-suspended 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. 'Empty' C43 cells also yield a current, and this is due to various chemicals that cells produce which may be involved in red-ox reactions with the working electrodes.

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. In experiments with working electrode potential of either E= 0.2V or E = 0.4V the OD600 of 'Empty' C43 cells drops at a more gentle slope than that of C43 with induced MtrCAB proteins; this indicates that cells are dying at a slower rate. The difference in the rate of decline in OD600 values might be due to either of three reasons; the first reason being that MtrCAB proteins make the membrane more susceptible to tears caused by electrostatic effects. The second reason is that the counter electrode potential is fluctuating to more extreme values of potentials, again rupturing the cells. The third reason is that because of the dehydrogenation of lactate, toxic quantities of NADH build up inside the cell, eventually killing it [LINK CARBON!!].

As one might have noticed is that only negative controls ('empty' C43 cells) and positive controls (Ajo-F strain) were tested; neither the uninduced Ajo-F strain nor C43 cells with the BBa_K1316011 (ccm cluster) and BBa_K1316012 (MtrCAB) bio-bricks were assayed. This is because the cloning of the BBa_K1316012 took so long that it was only finished two weeks before the wiki freeze. This indeed sounds like enough time to do the bio-reactor experiments; this was however not possible because the bioreactor broke, yielding us empty-handed. Since we did prove to that the bio-bricks were finally cloned, and these contained the exact same genes, promoters and Ribosome Binding sites as C640 and I5023, these results do The strain containing the two aforementioned bio-bricks was assayed with our micro-fluidics potentiostat system (Dropsens), as described below.

The experiments

Future work with a bio-reactor should include the C43 strain with our own biobricks

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)

3. 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)