Difference between revisions of "Part:BBa K2952015"

Revision as of 10:50, 11 October 2019

Translational unit for FDH_h overexpression

Usage and Biology

Formate dehydrogenase (FDH_h) facilitates this reaction: CO2 + NADH <-> HCOO- + NAD+. Formic acid is a chemical commodity and can be easily stored and transported, making it a stable form of hydrogen fuel. It can be converted into hydrogen gas by E.coli's native formate hydrogen lyase (Yoshida et al., 2005). This formate dehydrogenase has high binding affinities for NADH and CO2, and is expected to generate formate efficiently (Alissandratos et al., 2013).

Our modelling suggested that overexpression of FDH_h in E.coli should increase hydrogen production more than any other hydrogenase.

Figure 1: Hydrogen (H2) flux for various E. coli strains growing on different media with a single carbon source using flux balance analysis (FBA). Biomass growth rate was set as the objective, while the uptake rate for carbon sources was set to -20mmol/gDWh. This shows that FDH_h-expressing E. coli out-performs all other strains, while galactose was the best carbon source.

Assembly and Functionality

Figure 2: Assembled plasmid was digested with SacII, expected bands were 1.8kb and 3kb.

Translational unit for FDH_h overexpression was assembled using loop assembly. Successful assembly was confirmed using diagnostic digestion and sequencing.

His-tagged FDH_h was over-expressed in E. coli BL21DE3 using T7 promoter (BBa_I712074). Successful expression after induction with IPTG was confirmed by his-tag purification and subsequent SDS-PAGE.

Figure 3: FDH_h was purified using Ni-affinity resin. Supernatant (S/N), flow trough 1 and 2 (FT1/FT2), and eluted fractions 1 and 2 were run on a 10% SDS-PAGE gel. The expected size of our protein is 80.7kDa. The concentration of purified protein was determined using Bradford assay and was shown to be 1.8µM.

Activity was assayed through NADH oxidation at 37° and measuring absorbance at 340nm. This showed that recombinant FDH_h can be expressed in E. coli while still maintaining functionality.

Figure 4: NADH oxidation assay was carried out using purified FDH protein. 0.1M 6.8pH sodium phosphate, 0.2mM NADH, 0.1M sodium bicarbonate and 0.2µM FDH were mixed together, and the absorbance was measured at 340nm. Soluble protein fractions from FDH-expressing E.coli and WT E. coli were used as controls (0.5mg/ml of protein was added to the reaction mixture described above). All measurements were done in triplicates and averaged. This assay showed that recombinant FDH_h can be expressed in E.coli and retain functionality.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1299
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 133
Illegal NgoMIV site found at 526
1000
COMPATIBLE WITH RFC[1000]

@@ Line 49: / Line 49: @@
 ===Assembly and Functionality===
+[[File:fdh1.jpg|200px|thumb|left|Figure 2: Assembled plasmid was digested with SacII, expected bands were 1.8kb and 3kb.]]
 Translational unit for FDH_h overexpression was assembled using loop assembly. Successful assembly was confirmed using diagnostic digestion and sequencing.
 His-tagged FDH_h was over-expressed in E. coli BL21DE3 using T7 promoter (BBa_I712074). Successful expression after induction with IPTG was confirmed by his-tag purification and subsequent SDS-PAGE.
-[[File:SDS_PAGE_FDH.jpeg|600px|thumb|left|Figure 2: FDH_h was purified using Ni-affinity resin. Supernatant (S/N), flow trough 1 and 2 (FT1/FT2), and eluted fractions 1 and 2 were run on a 10% SDS-PAGE gel. The expected size of our protein is 80.7kDa. The concentration of purified protein was determined using Bradford assay and was shown to be 1.8µM.]]
+[[File:SDS_PAGE_FDH.jpeg|500px|thumb|right|Figure 3: FDH_h was purified using Ni-affinity resin. Supernatant (S/N), flow trough 1 and 2 (FT1/FT2), and eluted fractions 1 and 2 were run on a 10% SDS-PAGE gel. The expected size of our protein is 80.7kDa. The concentration of purified protein was determined using Bradford assay and was shown to be 1.8µM.]]
@@ Line 104: / Line 105: @@
+Activity was assayed through NADH oxidation at 37° and measuring absorbance at 340nm. This showed that recombinant FDH_h can be expressed in E. coli while still maintaining functionality.
+[[File:NADH_oxidation.jpeg|800px|thumb|left|Figure 4: NADH oxidation assay was carried out using purified FDH protein. 0.1M 6.8pH sodium phosphate, 0.2mM NADH, 0.1M sodium bicarbonate and 0.2µM FDH were mixed together, and the absorbance was measured at 340nm. Soluble protein fractions from FDH-expressing E.coli and WT E. coli were used as controls (0.5mg/ml of protein was added to the reaction mixture described above). All measurements were done in triplicates and averaged. This assay showed that recombinant FDH_h can be expressed in E.coli and retain functionality.]]
-Activity was assayed through NADH oxidation at 37° and measuring absorbance at 340nm. This showed that recombinant FDH_h can be expressed in E. coli while still maintaining functionality.
-[[File:NADH_oxidation.jpeg|800px|thumb|left|Figure 3: NADH oxidation assay was carried out using purified FDH protein. 0.1M 6.8pH sodium phosphate, 0.2mM NADH, 0.1M sodium bicarbonate and 0.2µM FDH were mixed together, and the absorbance was measured at 340nm. Soluble protein fractions from FDH-expressing E.coli and WT E. coli were used as controls (0.5mg/ml of protein was added to the reaction mixture described above). All measurements were done in triplicates and averaged. This assay showed that recombinant FDH_h can be expressed in E.coli and retain functionality.]]