Part:BBa_K1316012
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
Measurements of EET in Self-Constructed 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 favorable 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 redox 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.
Self-made bioreactor
Figure 4 shows a schematic representation of our bioreactor. The working electrode is made of a square piece of carbon cloth (0.031m2) [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 when 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 Standard Hydrogen Electrode (SHE)[1]. 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 are attached, and pictures of the individual components are shown in figures 4 and 5.
Metabolism and the source of electrons for the MtrCAB pathway
There is but a limited scope of substrates that are expected to be able to act as electron donors for the MtrCAB pathway, see Carbon Metabolism and Electron Transport which are: lactate, N-acetylglucosamine, formate, and hydrogen [4]. 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 figure 6:
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.
Measurement of current
In the first experiment we tried to roughly replicate the conditions as stated in the Jensen [1] article; the exact protocol for seeding the bioreactor can be found in the protocol for bioreactor here . Figure 7 represents the current in mA divided by the first OD600 measurement at the start of the experiment; figure 8 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, and cloning into biobrick vectors took a long time. Choosing a low copy number vector is crucial to the successful implementation of the pathway since the MtrCAB proteins become toxic when over-expressed. We implemented BBa_K1316012 (MtrCAB + T7 pLac) in a pSB3K3 vector which has a comparably low copy-number to the vector used in [1].
Figure 7A shows a significant difference in current measured between the empty C43 strain and the Ajo-F strain that was induced overnight at working electrode potential E = 0.2V. 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 7B 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 8. This drop in OD600 is again roughly correlated to the decrease in current, proving that current is produced by live cells. The reason that cells die at all when seeded in the bioreactor is probably due to the fact that they cant produce enough ATP to sustain their maintenance needs in terms of ATP production. Lactate is not a substrate that yields energy when catabolized anaerobically by bacteria.
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 or a combination 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.
As one might have noticed is that only negative controls ('empty' C43 cells) and the Ajo-F strain were tested; neither the uninduced Ajo-F strain nor C43 cells with the BioBricks in an iGEM (pSB) backbone 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. The strain containing the two aforementioned bio-bricks in pSB backbone was assayed with our micro-fluidics potentiostat system (with the Dropsens electrode), as described below.
Conclusion and future experiments
In conclusion our biobricks themselves worked as expected yielding a current when a positive potential is applied to a working electrode of a potentiostat, and the current increases at larger working electrode potentials. Due to time-limitations we did not assay the inducability of the pathway, which would be crucial in terms of making a biosensor. Future work with a bio-reactor should include the C43 strain with the biobricks in biobrick (pSB) vector. Also experiments where the cells are seeded into the bioreactor before being induced, and induction happening after seeding would aid the proof of the use of these biobricks as a biosensor output. Furthermore different media supplemented with various electron donors such as formate would be interesting, to see what electron donor/medium combo yields the best results in terms of current and cell-death. Also media with a mix of a C-source and an electron donor would be very interesting since the cells would than potentially grow, while also still fuelling the MtrCAB pathway with electrons.
Microfluidic Device for Measurements of Current
The Microfluidic device described in the Microfluidics section, was used to attempt to characterize the BBa_k1316011 and BBa_k1316012 BioBricks. These constructs had already been characterized using the bioreactor, so the goal of these experiments was to establish whether the system could be scaled down to microlitre volumes.
6 different analytes were tested in the device: the induced and uninduced Ajo-Franklin strains were used as positive controls, and empty E. coli chassis, and water were used as a negative controls. Finally, induced and uninduced strains containing the two BioBricks in question were tested. Water was flushed through the device between each test. These tests were all performed at 0.2V and 0.4V electrode potential, as was used in the bioreactor experiments.
At 0.2V, we see an exponential decrease in current after application of potential. This can be explained the a capacitance effect between the electrodes. All strains stabilize to a current approximately equal to zero - and less than that of pure water. It can be concluded that at the small volumes involved with the microfluidics device (33ul) it might well be that there are not enough cells to produce a measurable current. Reasons for the negative currents seen in the Ajo-Franklin strains remain unknown at this time.
Similarly, at 0.4V electrode potential, a capacitance effect is seen, followed by no significant current from any of the strains. The flat line of the uninduced Ajo-Franklin suggests an equipment malfunction and thus should be discounted.
To conclude, measurable outputs from microfluidic devices with our cells has not been possible, so further development, either in the biology, or a redesign of the device should be chosen - perhaps with an alternative solution to microfluidics. This being said, the use of our designed microfluidic device offered several advantages over standard benchtop techniques (such as the bioreactor), so it is a field worth pursuing for use in other applications to improve the ease and efficiency with which experiments can be carried out. Advantages of the microfluidics over the bioreactor coupled to a potentiostat are the quantity of medium including relatively expensive compounds such as delta-ALA (precursor for heme), which is far less in the microfluidic device. Also, microfluidics has potential for in-field measurements, where the bioreactor is not to be handled on unsteady grounds and without regular flushing in order to retain an anaerobic environment. We are urging upcoming teams to reevaluate and succesfully implement microfluidic devices combined with electrodes.
Conclusions
From our characterization experiments we can conclude that:
- The mtrCAB genes under control of the weakened T7lacO promoter were successfully BioBricked.
- The ccm genes under control of the pFAB640 promoter were successfully BioBricked.
- Expression of both our mtrCAB BioBrick and ccm BioBrick results in Extracellular Electron Transport.
For more interest on characterisation of the mtrCAB genes check Electron Transport characterisation wiki page !
References
1. http://en.wikipedia.org/wiki/Standard_hydrogen_electrode
2. 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)
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)
Improvement from team iGEM17_USTC
Mtr CAB is a protein complex located on the outer membrane of Shewanella originally, transferring electrons from the cityplasm to the outside of the bacteria. However, according to the lately research, we found that this Mtr CAB protein complex can also transport electrons into the cytoplasm from the electrode or other electron donors from the outside. This is the role Mtr CAB plays in our project. We introduce this protein complex into our engineered E.coli to transport electrons into the cytoplasm. Mtr CAB consists of three proteins, Mtr A, Mtr B, Mtr C. Mtr B is anchored onto the outer membrane. With its ß barrel conformation, it can help to locate the Mtr A and Mtr C and increase the whole complex’s stability. Mtr A and Mtr C are the two protein that can actually transfer electrons with the heme attached into these two protein at the right position. MtrA is a 32-kD periplasmic decaheme cytochrome c, and MtrC is a 69-kD cell-surface-exposed. Electrons are collected by Mtr C then it will shuttle through this electron tunnel and go to Mtr A, then these electrons will be transferred to CymA on the inner membrane and final get to the NADH dehydrogenase through the electron transfer chain. With this protein complex, we can utilize the electrons from the electrode or some chemical compound outside of the bacteria can turn them into NADH when they get into the cytoplasm of our engineered bacteria, increasing the NADH’s concentration inside of the cytoplasm, which means their the reduction power ——the ability to synthesize, will be pump up. As we have mentioned, it’s the heme attached to the Mtr A and Mtr C that enable them to shuttle electrons. So these two protein belong to a protein family called cytochrome c.Introduction
The new part we have submitted is part BBa_K2242363.This year, we improve the function of part BBa_K1316012 from team iGEM14_TU_Delft-Leiden. In former iGEM teams, this part, or specifically this protein, are used to transfer intracellular electrons out to the electrode. In another word, it is a useful component if we want to build up a bio-anode. However, few iGEM teams, or even other researchers, have noticed that this protein, Mtr CAB can transfer electrons bidirectionally!! In our project, we successfully proved that Mtr CAB can enable our engineered bacteria to transfer extracellular electrons into the cytoplasm!
Function of Mtr CAB
In our project, Mtr CAB is the most important and fundamental proteins as it plays the role to transfer extracellular electrons into the cytoplasm through the membrane. To examine whether the Mtr protein complex has the function as we expected, we used our engineered strain pMC( strain co-expressed Mtr CAB and Ccm A-H) to construct a bio-cathode. We monitored the current of the bio-cathode to see whether there would be a higher current in the experiment group than the WT strain.
Here, first we did an bacteria PCR to monitor the maintenance of the recombinant plasmids(pM28 contains the mtr CAB’s gene and the pTBC contains the ccm A-H’s gene). As we can see in figure 11, we could confirm that the strain is fine to use.
Figure 11. Electrophoresis result of PCR of Mtr and Ccm
So we started our bio-cathode assay to examine our theory. The protocol of the bio-cathode assay can be found in the notebook part in our wiki, look it up if you want to know more details!! In figure 12, we can easily confirm that the Mtr CAB protein complex was mature, as the pellets were red in pMC group, no matter the strain had been induced or not. When we did it the first time, there was no significant difference of the current of the bio-cathode between WT and our strain pMC(data not shown). We speculated that it was because we did NOT have the starvation step when we first did it, which is to cultivate the bacteria in a minimal salts medium for a certain time, like 4 to 6 hours. Because we did NOT have this starvation step, although we already used PBS to wash the bacteria 2 to 3 times, those nutritions still contained inside of the bacteria, providing another electron source when we were running the bio-cathode. So when the cathode was given a certain voltage, the bacteria still wouldn’t take up the electrons from the electrode.
Figure 12. Bacteria sediments
(from left to right, WT, pMC not induced, pMC induced)
So we performed this bio-cathode assay for a second time, adding this starvation step into the protocol. In addition, after the starvation step, we used 1 mL of minimal salts medium to resuspend the bacteria and dropped it onto the graphite electrode to form a bio-film, which could help to make a better connection between the bacteria and the electrode, especially when we were using the Mtr pathway to transfer electrons. Here, in figure 13, you can see how we made this biofilm. 2 to 3 hours later, with a sufficient airflow in the laminar flow hood, the graphite electrode would dry up and form a great biofilm. With this biofilm, electrons could be transferred to the Mtr C protein directly from the electrode which can increase the efficiency of electron transferring. Then what we need to do was to construct this bio-cathode, put every part of this “toy” together and get the oxygen out of this container. Here in figure 14 is how we clear the oxygen out of the bio-cathode to create an anaerobic environment. Lastly, we connected the bio-cathode to the electric-chemical station to give a certain voltage to the cathode and monitor the current of the cathode as time went by as how figure 15 shows.
Figure 13. Preparation for bio-film
Figure 14. Preparation for reaction system
(to exclude oxygen out of the container)
Figure 15. Bio-cathode device
Figure 16 is the result of this experiment. From the figure, we can easily notice that the red line, which is the Mtr-induced group, had a 50% higher current than the other two group after the bio-cathode turned into a stable state . This could strongly prove that the engineered strain pMC can transfer electrons into the cytoplasm, which led to the increasing of the cathode-current. But there would be a chance that this difference between these 3 groups was just the background noise between this three cathode, resulting from the hardware’s varieties. So we added fumarate into the system to see whether there would be a cathode catalyzed current happened in the pMC group. That’s why there was a sharp increasing in the figure. When we added fumarate into the system, the electrons on the electrode finally found a way to leak to—— the fumarate. So there would be a strong electron flow when we added fumarate into the system. But after a short time we introduced this sudden change into the system, the current will become stable again, slowly climbing back to the current it was. However, the time it took to get back to stable state can be a strong evident to prove our assumption——our engineered E.coli can transfer extracellular electrons into the cytoplasm!! The red line’s curve happened after we added fumarate into the system is kind of a typical curve of cathode-catalyze-current!! So, with this result, the cathode’s current to time under a certain voltage, we can confidently say that the Mtr CAB system work!!
Figure 16. The current result of the bio-cathode.
In conclusion, the Mtr CAB system can really function as an electron pathway to transfer extracellular electrons into the cytoplasm, even though it’s expressed in E.coli, but not it’s origin host Shewanella.!! In another word, our conduction system can function as we expected, transferring those electrons from the electrode into the cytoplasm, which means our E.coli can transform itself like transformer from a normal form to a special form that can “eat” electrons!
Reference:
[1] Thomas, P. E., Ryan, D., & Levin, W. (1976). An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Analytical biochemistry, 75(1), 168-176.[2] Jensen, H. M. (2013). Engineering Escherichia coli for molecularly defined electron transfer to metal oxides and electrodes. University of California, Berkeley Sequence and Features
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