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Outer Membrane Porin OprF

Two extracellular electron transfer pathways have been identified in the reduction of graphene oxide (GO) by Shewanella oneidensis MR-1. These are indirect electron transfer, mediated by secreted electron shuttles, and direct extracellular electron transfer (DET) which involves direct contact with the extracellular material. To increase the number of electron shuttles exiting the cell into the extracellular space. Increased extracellular electron donors should increase the reduction rate by Shewanella oneidensis MR-1

Usage and Biology

Shewanella oneidensis MR-1 are gram-negative bacteria at the center of studies of microbial reduction due to their ability to transfer electrons extracellularly to reduce materials such as graphene oxide (GO)[1]. Such characteristics have made S. oneidensis MR-1 an organism of interest in microbial fuel cells for bioelectricity generation and potential applications in bioremediation[2]. Two extracellular electron transfer pathways have been identified in the reduction of GO by Shewanella oneidensis MR-1. These are indirect electron transfer, mediated by secreted electron shuttles, and direct extracellular electron transfer (DET) which involves direct contact with the extracellular material[3]. The oprf gene found in Pseudomonas aeruginosa encodes the outer membrane porin F which is a nonspecific porin that allows for the diffusion of various molecules across the outer membrane.[4] The expression of this porin in S. oneidensis MR-1 has been seen to increase the electron shuttles, flavins, in the extracellular space leading to an increase in power generation in microbial fuel cells.[3] Another study has shown that this expression results in increased biofilm formation due to increased membrane permeability which ultimately increased bioelectricity generation[5]. oprf is seen to play a role in both the indirect and direct pathways for electron transfer in S. oneidensis MR-1 through the increase in electron shuttles and the increase in contact with the terminal electron acceptor via increased biofilm production. It was thought that the expression of oprf in S. oneidensis MR-1 would lead to an increase in flavins and other electron shuttles in the extracellular space as well as potentially increase biofilm production. This in turn would result in an increased rate of reduction of GO.

We therefore, synthesized the oprf gene, obtained from the Pseudomonas aeruginosa genome , and inserted it into the kanamycin resistant vector pcD8 under the control of an IPTG-inducible promoter (Keitz lab, University of Texas Austin)[6] for expression in S. oneidensis MR-1.

Results

Optical density measurements (O.D.₆₀₀)

Previous studies have shown that the magnitude of reduction of graphene oxide can be measured using optical density measurements at a wavelength of 600 nm. It has also been shown that the presence of graphene oxide has no significant effect on the growth rate of S. oneidensis MR-1.[6] So, we utilized this technique to measure and compare the magnitude of reduction with our different transformed strains and the wildtype.

Expression of our genes in our IPTG-inducible vector, pcD8, was initially induced with 1.5mM IPTG. Immediately following induction, reduction of graphene oxide was carried out under constant shaking at 200 rpm, and the O.D.₆₀₀ was measured every hour for 48 hours for TSB only, graphene oxide only, graphene oxide and TSB only, and graphene oxide and TSB each with oprf and the wildtype. Here, the TSB only , graphene oxide only, and graphene oxide and TSB only are our negative controls, and wild type MR-1 is the positive control to which all our transformed strains are compared. The O.D.₆₀₀ measurements obtained from these experiments reflect both bacterial absorbance as well as reduced graphene oxide absorbance. So, the absolute O.D.₆₀₀ due to reduced graphene oxide is obtained by correcting for O.D.₆₀₀ of graphene oxide and TSB only and bacteria and TSB only.

Figure 1: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to oprf (green) for the reduction period. Here, time zero reflects the start of induction with 1.5mM IPTG.

Figure 1 shows the microbial reduction of graphene oxide with the transformed strain, oprf. It represents the combined O.D.₆₀₀ measurements for the reduced graphene oxide absorbance and bacterial absorbance. The figure shows that transformation of S. oneidensis MR-1 with oprf still allows for reduction following a lag from the initiation of reduction as compared to the wildtype and pcD8. However, at its steepest point, the O.D.₆₀₀ curve, which signifies the increase in reduction, is comparable to that of the wildtype and the empty vector, pcD8.

Figure 2: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (dark gray) compared to oprf (light purple) over a 48-hour period. Here, time zero reflects the start of induction with 1.5mM IPTG.

Figure 3: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to oprf (green)adjusting for the O.D.₆₀₀ values due to bacterial growth. Here, time zero reflects the start of induction with 1.5mM IPTG.

The bacterial growth over the 48-hour period was measured by means of O.D.₆₀₀ to determine the impact of oprf expression on bacterial growth. Figure 2 shows that bacteria expressing the oprf gene (light purple) had a delay in growth for the first 24 hours as compared to the wildtype (dark gray) and the other expressed genes. Beyond that point, the bacterial growth evens out to be about the same as the wildtype and pcD8.

To get the absolute O.D.₆₀₀, representative of the reduced graphene oxide content in the microbial reduction, the measured O.D.₆₀₀ were corrected for the O.D.₆₀₀ for bacterial growth in TSB only. Figure 3 shows that after correction for bacterial growth oprf , at the steepest point on its curve, had a comparable rate of reduction with the wildtype and pcD8. The lag in reduction with oprf may be due to its initial delay in growth when compared to the other strains.

Since oprf showed slower growth than wild-type MR-1 (Figure 2), we hypothesized that the induced gene may have been slightly toxic to the cells. We then observed the growth curve when the concentration of IPTG was decreased to 1.0mM to reduce the amount of oprf expression.

Figure 4: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to oprf (green). Here, time zero reflects the start of induction with 1.0mM IPTG.

Figure 5: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (dark gray) compared to oprf (light purple) over a 48-hour period. Here, time zero reflects the start of induction with 1.0mM IPTG.

Figure 6: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to oprf (green) adjusting for the O.D.₆₀₀ values due to bacterial growth. Here, time zero reflects the start of induction with 1.0mM IPTG.

Figure 4 shows that with 1.0mM IPTG induction, microbial reduction of graphene oxide with the transformed strain oprf still had the initial lag in reduction but within its steepest region, its reduction curve was comparable to wild-type MR-1 and the empty vector, pcD8.

Figure 5 shows that there was no significant alteration in the growth for oprf for induction with 1.0mM IPTG as compared with 1.5mM IPTG (Figure 2).

Figure 6 shows that with the correction for bacterial growth, microbial reduction with oprf with 1.0mM IPTG induction at the steepest region of its curve had a greater rate of reduction as compared to the wildtype and pcD8.

We carried out an induction time of 5 hours prior to reduction to allow oprf to already have the porin production fully active before we began the reduction reactions expression. This was carried out with either 1.5mM or 0.75mM of IPTG under the same conditions of shaking at 200 rpm.

Figure 7: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to oprf (deep blue) for the reduction period. Here, reduction was initiated following a 5 hour induction with 1.5mM IPTG (5hr induction)

From Figure 7, the microbial reduction of graphene oxide with the transformed strain oprf shows a greater rate of reduction to that of the wildtype without any corrections to the O.D.₆₀₀ measurements for bacterial growth. It also shows a comparable rate of reduction to that of pcD8.

Figure 8: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (red) compared to oprf (green) for the 48-hour period. Here, reduction was initiated following a 5 hour induction with 1.5mM IPTG (5hr induction)

The bacterial growth measured by O.D.₆₀₀ in Figure 8 shows that bacteria expressing the oprf gene (black) still had less overall growth over the 48-hour period and more variability in its growth curve as compared to the wildtype and the other expressed genes.

Figure 9: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to oprf (deep blue) adjusting for the O.D.600 values due to bacterial growth. Here, reduction was initiated following a 5 hour induction with 1.5mM IPTG (5hr induction)

After corrections for bacterial growth, Figure 9 shows that oprf had the fastest rate of microbial reduction as compared to the wildtype and pcD8.

Figure 10: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to oprf (green) for the reduction period. Here, reduction was initiated following a 5 hour induction with 0.75mM IPTG (5hr induction).

Figure 10 shows that the microbial reduction of graphene oxide with the transformed strain oprf at 0.75mM IPTG 5 hr induction has a greater rate of reduction than that of the wildtype and pcD8 without any corrections to the O.D.₆₀₀ measurements for bacterial growth.

Figure 11: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (red) compared to oprf (light blue) for the 48-hour period. Here, reduction was initiated following a 5 hour induction with 0.75mM IPTG (5hr induction).

Figure 11, shows that the delay in growth in oprf (green) with 0.75mM IPTG is decreased from that seen with 1.5mM IPTG (Figure 8). However, even with the concentration of IPTG reduced to half from 1.5mM, the growth curve of oprf still lags behind the wildtype and pcD8.

Figure 12: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to oprf (green) adjusting for the O.D.₆₀₀ values due to bacterial growth. Here, reduction was initiated following a 5 hour induction with 0.75mM IPTG (5hr induction).

Figure 11, shows that the delay in growth in oprf (green) with 0.75mM IPTG is decreased from that seen with 1.5mM IPTG (Figure 8). However, even with the concentration of IPTG reduced to half from 1.5mM, the growth curve of oprf still lags behind the wildtype and pcD8.

The growth curves of oprf show stochastic patterns for each of the IPTG conditions (Figure 2,5,8,11). It is thought that such patterns are observed due to the production of biofilm where increased biofilm production may cause bacteria to adhere to the sides of the plate wells, reducing the amount of oprf cells in the TSB for O.D.₆₀₀ measurements.

The slopes at the steepest point on the curves were obtained for each induction condition to give the maximum reduction rate measured during the reduction period, and this was compared to that of wild-type MR-1.

Figure 13: Maximum rates of the bacterial reduction curves adjusted for bacterial growth at the IPTG induction conditions of 1.5mM and 1.0 mM IPTG with 0hr induction and 1.5mM and 0.75mM IPTG with 5 hour induction.

The results show that after a 5hr induction with 1.5mM IPTG as well as 0.75mM IPTG, oprf has a faster rate of reduction as compared to wild-type MR-1 (Figure 9,12). Figure 13 shows that the maximum rate obtained for oprf at these conditions were 8.836 hr^-1 and 8.468 hr^-1, respectively, whereas the wildtype had a rate of 7.732 hr^-1. There are comparable reduction rates with 1.5mM and 1.0mM IPTG for reduction immediately following induction (Figure 3,6). The reduction with oprf appears to be more similar to wild-type MR-1 at the lower IPTG concentration, 1.0mM (Figure 7).

The negative controls (TSB only, GO only, and GO and TSB only) show an insignificant change in O.D.₆₀₀ over time indicating that the bacteria are responsible for the increase in O.D.₆₀₀, that is, reduction.

Raman spectroscopy

Raman spectroscopy was carried out to investigate the amount of carbon-carbon single bonds of the reduced graphene oxide produced by oprf over the 48 hour period. Sequence and Features

References

[1] Wang, G.; Qian, F.; Saltikov, C. W.; Jiao, Y.; Li, Y. Microbial Reduction of Graphene Oxide by Shewanella. Nano Research 2011, 4, 563–570.
[2] Schwalb, C.; Chapman, S. K.; Reid, G. A. The Tetraheme Cytochrome Cyma Is Required for Anaerobic Respiration with Dimethyl Sulfoxide and Nitrite in Shewanella Oneidensis. Biochemistry 2003, 42, 9491–9497.
[3] Lin, T.; Ding, W.; Sun, L.; Wang, L.; Liu, C.-G.; Song, H. Engineered Shewanella Oneidensis-Reduced Graphene Oxide Biohybrid with Enhanced Biosynthesis and Transport of Flavins Enabled a Highest Bioelectricity Output in Microbial Fuel Cells.

[4] Sugawara, E.; Nestorovich, E. M.; Bezrukov, S. M.; Nikaido, H. Pseudomonas Aeruginosa Porin Oprf Exists in Two Different Conformations. Journal of Biological Chemistry 2006, 281, 16220–16229.
[5] Lin, T.; Bai, X.; Hu, Y.; Li, B.; Yuan, Y. J.; Song, H.; Yang, Y.; Wang, J. Synthetic Saccharomyces Cerevisiae ‐ Shewanella Oneidensis Consortium Enables Glucose‐Fed High‐Performance Microbial Fuel Cell. AIChE Journal 2016, 63, 1830–1838.
[6] Dundas, C. M.; Walker, D. J. F.; Keitz, B. K. Tuning Extracellular Electron Transfer by Shewanella Oneidensis Using Transcriptional Logic Gates. ACS Synthetic Biology 2020, 9, 2301–2315.