Team:Bielefeld-Germany/Project/GldA
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- | [[Image:IGEM_Bielefeld_Table7_ExpDesign_NADH.jpg|300px|thumb|left|<p align="justify"> '''Table 3: Experimental design of the glycerol dependent [[Team:Bielefeld-Germany/Labjournal/Molecular#NADH-Assay|NADH-assay]]. Supplementation of different amounts of glycerol to | + | [[Image:IGEM_Bielefeld_Table7_ExpDesign_NADH.jpg|300px|thumb|left|<p align="justify"> '''Table 3: Experimental design of the glycerol dependent [[Team:Bielefeld-Germany/Labjournal/Molecular#NADH-Assay|NADH-assay]]. Supplementation of different amounts of glycerol to [[Team:Bielefeld-Germany/Labjournal/Molecular#LB medium|LB medium]] should show the effect of glycerol on NADH overproduction. '''</p>]] |
Revision as of 20:55, 30 September 2013
GldA
Glycerol dehydrogenase GldA - Overview
The Gene GldA encodes glycerol dehydrogenase from Escherichicha coli. The overexpression of glycerol dehydrogenase in E. coli can be used as a way for endogenous mediator production. Many derivates of glycerol dehydrogenase are small, water-soluble redoxmolecules, which have mediator properties. In enzymology, a glycerol dehydrogenase (EC 1.1.1.6) is an enzyme belonging to the family of oxireductases that catalyzes the chemical reaction: Glycerol + NAD+ <--> Glycerone + NADH + H+ with NADH as the main endogenous mediator.
Theory
- Theory for GldA glycerol dehydrogenase.
Genetic Approach
- The GldA gene from Escherichia coli was cloned and heterologously expressed in E. coli KRX under the control of different promoters (Table 1).
Results
- Upon the expression of the glycerol dehydrogenase, the endogenous mediator production of Escherichia coli was measured. SDS-PAGE combined with MALDI-TOF MS/MS and different NADH-assays characterize GldA BioBrick <bbpart>BBa_K1172201</bbpart>.
SDS-PAGE and MALDI-TOF
- SDS-PAGE shows protein Glycerol dehydrogenase at expected size of 40 kDa. In contrast to Escherichia coli KRX Wildtyp, weak Anderson promoter (<bbpart>BBa_K1172205</bbpart>) shows only a slightly stronger band, whereas T7 (<bbpart>BBa_K1172203</bbpart>) and Lac (<bbpart>BBa_K1172204</bbpart>) promotor show a strong band, which is equated with a strong expression and overproduction of GldA.
- Furthermore we were able to identify the overexpressed glycerol dehydrogenase (Figure. 2) with MALDI-TOF MS/MS.
- Tryptic digest of the gel lane for analysis with MALDI-TOF could examine the glycerol dehydrogenase with a Mascot Score of 266 against Escherichia coli database.
NADH-Assays
- An overproduction of the glycerol dehydrogenase results of course in an overproduction of products from glycerol dehydrogenase. Glycerol dehydrogenase produces several mediators because of its broad substrate specificity. GldA is involved in several metabolism pathways, for example in the glycerol metabolism, which converts glycerol and NAD+ to Glycerone, NADH and H+. NADH is a small, water-soluble redoxmolecule, which seems to be a great mediator for Microbial Fuel Cells. An overexpression of glycerol dehydrogenase leads to an overexpression of NADH and therefore to an endogenous mediator production. Furthermore GldA seems to be very interesting in combination with our preferred MFC carbon source glycerol. Thus, it is essential to get reliable data on NADH overproduction to see how many electron shuttles can be available and how efficient electron shuttle-mediated electron transfer (EET) will be.
- We could observe an enhanced NADH production for Escherichia coli KRX with pSB1C3 and <bbpart>BBa_K1172203</bbpart>, <bbpart>BBa_K1172204</bbpart> and <bbpart>BBa_K1172205</bbpart> with increasing promotor strength and in comparison with Escherichia coli KRX Wildtyp. (Table 2 and Figure 3)
- Compared with Escherichia coli KRX Wildtyp, Escherichia coli with GldA and T7 promotor (<bbpart>BBa_K1172203</bbpart>) shows 1,7 μM NADH overproduction and GldA with Lac promotor (<bbpart>BBa_K1172204</bbpart>) shows 0,2 μM NADH overproduction. The GldA expression by the Anderson promotor (<bbpart>BBa_K1172205</bbpart>) is too weak for an efficient NADH overproduction.
- Thus, E. coli with GldA and T7 promotor (<bbpart>BBa_K1172203</bbpart>) is used for all further tests, because this strain shows by far the best NADH production rate.
- Former MFC tests showed, that glycerol is the best carbon source for Escherichia coli in our Microbial Fuel Cell. Therefore it is important to see, which effect an increased glycerol concentration on the mediator production has. To test these parameters, LB medium was supplemented with different amounts of glycerol. (Table 3)
- E.coli grown in glycerol supplemented medium shows great enhanced NADH production in contrast to Escherichia coli KRX Wildtyp and also in contrast to Escherichia coli KRX with <bbpart>BBa_K1172203</bbpart> and no further NADH supplementation. (Table 4 and Figure 4)
- Glycerol dependent NADH-assay shows a quiet good NADH overproduction for <bbpart>BBa_K1172203</bbpart> with supplementation of glycerol. It was possible to increase NADH production up to 270 % in comparison to Escherichia coli KRX Wildtyp and up to 100 % in comparison to Escherichia coli KRX with <bbpart>BBa_K1172203</bbpart> and without glycerol supplementation. However, it is relatively unimportant how much glycerol is supplemented to the medium. However, this growth characteristics is initially only valid for LB medium glycerol supplementation.
- With former NADH-assays we only observed the effects of GldA on the intracellular NADH concentration of E. coli. To investigate additionally extracellular NADH concentration, we tested Escherichia coli KRX Wildtyp strain and E. coli KRX with pSB1C3 and <bbpart>BBa_K1172203</bbpart> by cultivation with different glycerol concentrations in M9 medium. (Table 5)
- Cultivating Escherichia coli <bbpart>BBa_K1172203</bbpart> with increasing glycerol concentration of M9 medium shows enhanced intracellular and also enhanced extracellular NADH concentration in contrast to Escherichia coli KRX Wildtyp. (Table 6 and Figure 5)
- In addition to previously observed increase in intracellular NADH concentration, we were able to show an enhanced extracellular NADH concentration. Heterologous expression of GldA <bbpart>BBa_K1172203</bbpart> in E. coli seems to be an appropriate way for endogenous mediator production. Furthermore glycerol could be confirmed as a suitable substrate for the NADH production.
- Intracellular NADH concentration increases up to 223 % in comparison to Escherichia coli KRX Wildtyp with increasing promotor strength. Besides it is very interesting to see that extracellular NADH concentration is 7 times higher than the intracellular concentration for all strains. Heterologous expression of GldA <bbpart>BBa_K1172203</bbpart> in E. coli causes a 2.5 times higher extracellular NADH concentration compared with Escherichia coli KRX Wildtyp.
- These data show that NADH is an adequate mediator for Microbial Fuel Cells. NADH can be transported across the cell membrane which is indicated by much higher extracellular NADH concentration and thus allow NADH-mediated electron transfer (EET).
Conclusion
- Mediators are essential for the use of Escherichia coli in Microbial Fuel Cells. The main advantage of improving MFCs is to enhance kinetics of the electron transfer between the bacterial cells and the fuel cell anode. In order to decrease the usage of expensive and toxic synthetic mediators (exogenous mediators like methylene blue oder neutral red), we produced the endogenous mediator NADH by overexpression of glycerol dehydrogenase.
- Looking at our project in its entirety, the overproduction of glycerol dehydrogenase seems to be a great way for endogenous mediator production. The use of glycerol as our main carbon source for Escherichia coli in the MFC further enhances the efficiency of NADH production. We can show a 270 % higher intracellular NADH concentration (4 μM) for Escherichia coli KRX with GldA plasmid and 170 % higher extracellular NADH concentration (24 μM) in contrast to Escherichia coli KRX Wildtyp.
- The heterologous expression of GldA is a great genetic strategy to optimize mediator production as well as electricity generation in Microbial Fuel Cells. The most suitable and efficient GldA device for Escherichia coli is a combination with Rhamnose inducible T7 promotor (<bbpart>BBa_K1172203</bbpart>).
References
- Xiang K, Qiao Y, Ching CB, Li CM (2009) GldA overexpressing-engineered E. coli as superior electrocatalyst for microbial fuel cells. [http://www.sciencedirect.com/science/article/pii/S1388248109002835| Electrochemistry Communications 11: 1593–1595].
- Zhang T, Cui C, Chen S, Ai X, Yang H, Shen P, Peng Z (2006) A novel mediatorless microbial fuel cell based on direct biocatalysis of Escherichia coli. [http://pubs.rsc.org/en/content/articlehtml/2006/cc/b600876c| Chem Commun 11: 2257–2259].
- Mulichak AM (2005) Crystal structure of glycerol dehydrogenase. [http://www.rcsb.org/pdb/images/1ta9_bio_r_500.jpg | Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Band (PDB)].