Re-suspended DNA (constitutive promoters) from iGEM Distribution kit and transformed them into DH5α strain.
Growth was found on the negative control plates so re-transformed DNA
Inoculated of transformed cells.
Pilot Experiment
Made stocks of our reagents Glucose Stock for 0.005 g/mL, Glucose Oxidase Stock for 0.0025 g/mL, HRP Stock for 250 μL/mL, ABTS for 0.0025 g/mL
Inducible Gene Switch
Performed miniprep for overnight cultures
Prepared fresh batch of DH5α chemical competent cells
Restriction enzyme digest of constitutive promoters and control(pUC19) for validation with EcoRI and PstI
Pilot Experiment
Still awaiting the arrival of the BMG spectrophotometer
Inducible Gene Switch
Prepared DH5α chemical competent cells
Prepared Chloramphenicol antibiotic stock
Restreaked alcA 1 (BBa_K678001) and spispink(BBa_K1033923 pink chromoprotein)
Pilot Experiment
We began by preparing master mix containing three reagents glucose oxidase, HRP and ABTS (table 1)
Final reagent (μg/ml)
Glucose oxidase
60
HRP
60
ABTS
100
Table 1. Master mix
Six different glucose concentrations were then made as depicted in table 2
Final glucose concentration (μg/ml)
0.50
1.00
.25
1.50
1.75
2.00
Table 2. Glucose concentration
Protocol
50 μl of glucose solution was added into each well. Samples were run in triplicates
Tube containing master mix was placed into spectrophotometer.
Spectrophotometer was then used to measure absorbance of our green coloured product-oxidised ABTS at 420 nm. Absorbance values were taken every 2 sec for 3 min once 150 ul of master mix was added into each well.
Results
The rate of reaction did not level off and poor colour intensity was observed.
We repeated the same experiment we did earlier this week. However instead of measuring absorbance for 3 min absorbance was measured every 2 sec for a 5 min period.
Results - Reaction rate did level off after measuring absorbance for 5 min. However, colour intensity was still very poor.
