Team:Victoria Wellington/Parts
Team
Victoria
Parts
Introduction
For this project we were interested in designing and characterising parts that can break the carbon-carbon bonds of mesoxalic acid, oxalic acid and formic acid. This is because, these three molecules cannot be oxidized but the small molecule TEMPO-NH2, preventing the full oxidation of glycerol to CO2. To rearrange mesoxalic acid and oxalic acid, we have designed the part OxDc. To oxidize formic acid we have designed two parts, mtFDH and cbFDH, with the goal of determining which part has the higher optimal pH, and so works better with TEMPO-NH2.
Parts Table
name
biobrick
type
designer
length
OxDc
BBa_K3155000
Basic
Mitch, Alex and Ethan
1158 bp
cbFDH
BBa_K3155001
Basic
Mitch, Alex and Ethan
1113 bp
mtFDH
BBa_K3155002
Basic
Mitch, Alex and Ethan
1101 bp
Characterisation
All kinetic assays used the same reaction conditions: pH =7.5, T = 30℃, [NAD+] = 10mM, in 100mM phosphate buffer
Conditions - T = 30℃, [NAD+] = 10mM, [formate] = 200mM in 100mM phosphate buffer(s).
Notes - cbFDH had been idle in freezer for a couple of months at this stage, while mtFDH was newly expressed. Additionally, mtFDH activity values inexplicably do not align with kinetic assays (see results section).
It was noted that activity in this assay did not align with those values observed for the pH assay. Another assay was conducted (using a new method, where absorbance was followed continuously).
This model was found to have a better fit and so was selected for calculation of kinetic parameters of mtFDH
Credit - Model fitting (for kinetics) was done using the drc package in R. All other plots were constructed in R with default packages.