Team:Bielefeld-Germany/Project/GldA
From 2013.igem.org
GldA
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
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References
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