Difference between revisions of "Part:BBa K3724008"
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The bacterial growth measured by O.D.<sub>600</sub> (Figure 9) shows that bacteria expressing the ydeh gene (brown) 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. There is still a delay, albeit a decreased one, in the growth of ribf (yellow) and oprf (blue). | The bacterial growth measured by O.D.<sub>600</sub> (Figure 9) shows that bacteria expressing the ydeh gene (brown) 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. There is still a delay, albeit a decreased one, in the growth of ribf (yellow) and oprf (blue). | ||
| − | [[File:T--Rochester--1.5mM_withbacteria5hr.png|450px|thumb|right|Figure | + | [[File:T--Rochester--1.5mM_withbacteria5hr.png|450px|thumb|right|Figure 7: O.D.<sub>600</sub> of microbial reduction with wild-type MR-1 (deep purple) compared to the transformed strains cyma, oprf, pcD8 (empty vector), ydeh, ribf and riboflavin cluster adjusting for the O.D.<sub>600</sub> values due to bacterial growth. Here, time zero reflects the start of induction with 1.5mM IPTG.]] |
| − | [[File:T--Rochester--Bacterialgrowth_1.5mM_5hr.png|450px|thumb|right|Figure | + | [[File:T--Rochester--Bacterialgrowth_1.5mM_5hr.png|450px|thumb|right|Figure 8: O.D.600 of microbial reduction with wild-type MR-1 (deep purple) compared to the transformed strains cyma, oprf, pcD8 (empty vector), ydeh, ribf and riboflavin cluster adjusting for the O.D.600 values due to bacterial growth. Here, time zero reflects the start of induction with 1.5mM IPTG.]] |
Figure 12, shows that the delay in growth in ydeh (green), oprf (blue) and ribf (yellow) with 0.75mM IPTG is decreased from that seen with 1.5mM IPTG (Figure 9). However, even with the concentration of IPTG reduced to half from 1.5mM, the growth curves of the previously mentioned genes still lag behind the wildtype and pcD8. | Figure 12, shows that the delay in growth in ydeh (green), oprf (blue) and ribf (yellow) with 0.75mM IPTG is decreased from that seen with 1.5mM IPTG (Figure 9). However, even with the concentration of IPTG reduced to half from 1.5mM, the growth curves of the previously mentioned genes still lag behind the wildtype and pcD8. | ||
Revision as of 06:31, 21 October 2021
Diguanylate cyclase YdeH
Functional Parameters
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 graphene oxide and TSB only and bacteria and TSB only.
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.
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 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).
[[File:T--Rochester--Bacterialgrowth_1mM.png|450px|thumb|right|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.
The results showed that ydeh had comparable reduction rates to wild-type MR-1 and the empty vector, pcD8 for induction with 1.5mM IPTG (Figure 3) and 1.0mM induction (Figure 6).
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 expression. This was carried out with either 1.5mM or 0.75mM of IPTG under the same conditions of shaking at 200 rpm (Figure 7,10).
The bacterial growth measured by O.D.600 (Figure 9) shows that bacteria expressing the ydeh gene (brown) 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. There is still a delay, albeit a decreased one, in the growth of ribf (yellow) and oprf (blue).
Figure 12, shows that the delay in growth in ydeh (green), oprf (blue) and ribf (yellow) with 0.75mM IPTG is decreased from that seen with 1.5mM IPTG (Figure 9). However, even with the concentration of IPTG reduced to half from 1.5mM, the growth curves of the previously mentioned genes still lag behind the wildtype and pcD8.
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.
These results show that after a 5hr induction with 1.5mM IPTG as well as 0.75mM IPTG, ydeh had the fastest rate of reduction as compared to the wild-type MR-1 and the other transformed strains (Figure 10,13). Figure 14 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 for ydeh with 1.5mM and 1.0mM IPTG for reduction immediately following induction (Figure 4,7). The reduction with ydeh appears to be more similar to wild-type MR-1 at the lower IPTG concentration, 1.0mM (Figure 7).
The growth curves of ydeh
All the IPTG conditions show that the riboflavin cluster and cyma have reduction rates comparable to each other, the wildtype, and pcD8, especially at 5hr induction with 1.5mM (Figure 10). oprf and ribf are seen to lag behind in reduction at all IPTG conditions, potentially due to the initial lag in their growth phase. The maximum reduction rates for riboflavin cluster, oprf, and ribf are greater than that of the wildtype for 1.0mM IPTG 0 hr induction. At 1.5mM IPTG 0hr induction the just riboflavin cluster and ribF also have maximum reduction rates greater than the wildtype (Figure 14).
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.
Overall, transforming S. oneidensis MR-1 with our inserts allowed for reduction of graphene oxide, where ydeH was shown to have the greatest rate of reduction, exceeding that of wild-type MR-1.
With these results, Raman spectroscopy was carried out to investigate the amount of carbon-carbon single bonds of the reduced graphene oxide produced by each of the transformed strains over the 48 hour period.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 10
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 7
Illegal BsaI.rc site found at 910
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.