Part:BBa_K3724008
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 signalling 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.
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.
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