Difference between revisions of "Part:BBa K3724008"

Latest revision as of 04:33, 17 December 2021

Diguanylate cyclase YdeH

YdeH in Escherichia coli catalyzes the synthesis of Bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) from two molecules of Guanosine-5'-triphosphate (GTP).

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]. 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 [2]. Electron transfer via the DET pathway can be made more efficient by promoting biofilm formation. YdeH in Escherichia coli is a diguanylate cyclase which catalyzes the synthesis of Bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) from two molecules of Guanosine-5'-triphosphate (GTP). c-di-GMP is an intracellular signaling molecule that, if in high concentrations within the cell, helps in promoting bacterial biofilm production.[3] It has previously been seen that overexpression of ydeh in S. oneidensis MR-1 leads to an increase in bioelectricity generation and biofilm production [4]. An increase in bioelectricity generation signifies an increase in extracellular electron transfer so we thought that overexpressing this gene in S. oneidensis MR-1 would lead to increased extracellular electron transfer which in turn would lead to increased reduction of GO as compared to wild-type MR-1.The exact mechanism by which the electron transfer is increased has not been elucidated but it is thought that increased biofilm production increases point of contact with the extracellular material allowing for more transfer of electrons via outer membrane cytochrome proteins.

We therefore, synthesized the ydeh gene, optimized for S. oneidensis MR-1 , and inserted it into the kanamycin resistant vector, pcD8, under the control of an IPTG-inducible promoter (Keitz lab, University of Texas Austin) [5].

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 ydeh 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: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ydeh (deep blue) 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, ydeh. It shows that transformation of S. oneidensis MR-1 with ydeh still allows for reduction at a rate comparable to that of the wildtype. This figure represents the combined O.D.₆₀₀ measurements for the reduced graphene oxide absorbance and bacterial absorbance.

Figure 2: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (dark gray) compared to ydeh (black) 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 ydeh (deep blue)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 ydeh expression on bacterial growth. Figure 2 shows that bacteria expressing the ydeh gene (black) had less overall growth over the 48-hour period and showed a more variable growth curve as compared to the wildtype (dark gray) and the other expressed genes.

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 ydeh had a comparable rate of reduction with the wildtype where it appeared to be slightly slower.

Since ydeh 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 ydeh expression.

Figure 4: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ydeh (deep blue). 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 ydeh (black) 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 ydeh (deep blue) 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 ydeh had comparable reduction rates to wild-type MR-1 and the empty vector, pcD8. From the figure it is seen that the rate of reduction of ydeh without corrections for bacterial growth is slightly greater than that of the wildtype.

Figure 5 shows that there was no significant alteration in the growth for ydeh 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 ydeh with 1.0mM IPTG induction had a similar rate of reduction to the wildtype and a faster rate of reduction than pcD8 for the first 5 hours.

We carried out an induction time of 5 hours prior to reduction to allow ydeh to already have the biofilm production fully active before we began the reduction reactions. 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 ydeh (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 ydeh 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 ydeh (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 ydeh 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 ydeh (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 ydeh 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 ydeh (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 ydeh 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 ydeh (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 ydeh (light blue) 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 ydeh still lags behind the wildtype and pcD8.

Figure 12: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ydeh (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 12, shows that the microbial reduction of graphene oxide with the transformed strain ydeh at 0.75mM IPTG 5 hr induction has a greater rate of reduction than that of the wildtype and pcD8 after adjusting for the bacterial growth.

The growth curves of ydeh 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 ydeh 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, ydeh 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 ydeh 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 ydeh appears to be more similar to wild-type MR-1 at the lower IPTG concentration, 1.0mM (Figure 6).

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 ydeh 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.

Figure 14: D/G ratio for rGO produced under microbial conditions and chemical conditions.

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 ydeh resulted in a D/G ratio of 0.97 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.

Conclusion

We were able to successfully transform the E. coli diguanylate cyclase YdeH into S. oneidensis MR-1. This transformed strain showed faster rates of reduction with controlled expression of ydeh and also reduced the graphene oxide to levels expected with microbial reduction for S. oneidensis MR-1. This indicates that expression of ydeh in S. oneidensis MR-1 can decrease the time it takes for graphene oxide to be reduced to expected reduction levels for microbial reduction.

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] 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. Nano Energy 2018, 50, 639–648.
[3] Spangler, C.; Kaever, V.; Seifert, R. Interaction of the Diguanylate Cyclase Ydeh of Escherichia Coli with 2′,(3′)-Substituted Purine and Pyrimidine Nucleotides. Journal of Pharmacology and Experimental Therapeutics 2010, 336, 234–241.
[4] Liu, T.; Yu, Y.-Y.; Deng, X.-P.; Ng, C. K.; Cao, B.; Wang, J.-Y.; Rice, S. A.; Kjelleberg, S.; Song, H. Enhanced Shewanella Biofilm Promotes Bioelectricity Generation. Biotechnology and Bioengineering 2015, 112, 2051–2059.
[5] 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.
[6] 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.