Difference between revisions of "Part:BBa K3724011"
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| − | + | <i>oprf</i> in <i>Pseudomonas aeruginosa</i> encodes the nonspecific outer membrane protein F, oprF. | |
Revision as of 02:02, 22 October 2021
Outer Membrane Porin OprF
oprf in Pseudomonas aeruginosa encodes the nonspecific outer membrane protein F, oprF.
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.[7] 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 bacteria and TSB only.
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.
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 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.
From Figure 7, the microbial reduction of graphene oxide with the transformed strain oprf shows a comparable rate of reduction to that of the wildtype after its initial delay without any corrections to the O.D.600 measurements for bacterial growth. It also shows a comparable rate of reduction to that of pcD8.
The bacterial growth measured by O.D.600 in Figure 8 shows that bacteria expressing the oprf gene (light purple) still had the initial delay with the 5hr induction, however it is shorter as compared to the delay in the 1.5mM IPTG and 1.0mM IPTG 0hr induction (Figure 2,5). It also has a greater exponential increase in growth and exceeds the growth of the wildtype and pcD8 after 24 hours.
After corrections for bacterial growth, Figure 9 shows that oprf has a comparable rate of microbial reduction at the steepest region of its curve to the curves for wildtype and pcD8.
Figure 10 shows that the microbial reduction of graphene oxide with the transformed strain oprf at 0.75mM IPTG 5 hr induction has a comparable rate of reduction with that of the wildtype and pcD8 following its initial delay in reduction without any corrections to the O.D.600 measurements for bacterial growth.
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, shows that the microbial reduction of graphene oxide with the transformed strain oprf at 0.75mM IPTG 5 hr induction has a comparable rate of reduction with that of the wildtype and pcD8 following its initial delay in reduction after adjusting for the bacterial growth.
The growth curves of oprf show an initial delay with each of the IPTG conditions (Figure 2,5,8,11). However, with lower concentrations of IPTG, there was a shorter delay in bacterial growth which may indicate that over-expression of oprf may have some toxic effect on S. oneidensis MR-1.
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.
The results show that after a 0hr induction with 1.mM IPTG, the max rate of oprf was 9.969 hr-1 which was greater than that of the wildtype (8.936 hr-1). After 0 hr induction with 1.5mM IPTG, oprf had a max rate of 7.964 hr-1 which was comparable to the max rate of the wildtype of 8.936 hr-1. For the 5 hr induction with 1.5mM IPTG and 1.0mM IPTG, the max rates were still comparable to the wildtype and pcD8 albeit, slightly slower.
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 which is the measure of reduction of graphene oxide in these experiments.
Raman spectroscopy
Raman spectroscopy was carried out to investigate the amount of carbon-carbon single bonds of the reduced graphene oxide produced by oprf after the 48 hour period. We primarily relied on the D/G ratio, which is the intensity of the D peak divided by the intensity of the G peak. The D peak is associated with double-bonded carbon, and the G peak is associated with single-bonded carbon.
We used chemically reduced GO as our positive control as chemical reduction with ascorbic acid results in a much greater degree of reduction than seen in microbial reduction. We also had two negative controls: GO only and GO and TSB media only.
After reduction, we compared the D/G ratios to determine how well our transformed strain had done in reducing the number of Sp2 carbons.
As Figure 14 shows, the chemically reduced GO was by far the most reduced, with an average D/G ratio of around 1.2. The microbial reduction with oprf resulted in a D/G ratio of 0.98 which is close to the D/G ratio of wildtype. This demonstrates that our part worked as intended and was able to reduce the GO to expected levels of reduction during the reduction period.
Conclusion
We were able to successfully transform the P. aeruginosa outer membrane porin F, oprF, into S. oneidensis MR-1. This transformed strain showed greater maximum reduction rates with controlled expression and also reduced the graphene oxide to levels expected with microbial reduction for S. oneidensis MR-1. This indicates that expression of oprf in S. oneidensis MR-1 ,operating at its maximum rate of reduction, may decrease the time it takes for graphene oxide to be reduced to expected reduction levels for microbial reduction.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 7
Illegal BsaI.rc site found at 1072
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.
[7] Lehner, B. A.; Janssen, V. A.; Spiesz, E. M.; Benz, D.; Brouns, S. J.; Meyer, A. S.; van der Zant, H. S. Creation of Conductive Graphene Materials by Bacterial Reduction Using Shewanella Oneidensis. ChemistryOpen 2019, 8, 888–895.