Author: Nicolas Kylilis PhD
Automated data analysis workflow for the calculation of gene expression rate
Suitable for time-course fluorescence assays on microplate-reader for bacterial cells
Data analysis will only take place correctly if the following microplate layout is applied.
Column 1: Media blank
Column 2: Control cells for growth rate and autofluorescence
Column 3-12: Sample experimental conditions
Rows A-H: Replicates
- Growth rate calculation:
Based on formula: λ=[ln(OD_t2)- ln(OD_t1)]/dt
Where t = data collection time-point
Data processing steps for growth rate:
--Subtraction of media absorbance value from [OD data]
--ln [OD data]
--dy = mean(ln[ODt0], ln[ODt1], ln[ODt2]) - mean(ln[ODt-1], ln[ODt0], ln[ODt1])
--dt = t2-t1
--lambda = dy/dt
- Gene expression rate calculation:
Based on formula:
Protein production rate=number of cells*(Gene expression rate-[Protein]*dilution rate)
Assumptions: Max growth rate timepoint represents steady state of balance growth where FP production rate per cell = 0 (gene expression and diluation cancel each other out)
Data processing steps for Fluorescence/cell:
--Subtraction of media autofluorescence from [fluorescence data]
--Subtraction of media absorbance from [Absorbance data]
--Fluorescence per cell = [corrected Fluorescence data] / [corrected Absorbance data]
Data processing steps for Gene expression rate:
--CorrectedFI/cell = FI/cellmax_gr – FI/cellautfluorescence_max_gr
--Gene expression rate = CorrectedFI/cell * max growth rate
Data processing steps for Steady state assumption check by calculating FP production rate per cell (should be = 0 at macx growth rate):
--dP = mean(FI/cellt0, FI/cellt-1, FI/cellt1) - mean(FI/cellt-1, FI/cellt0, FI/cellt-2)
--dt = t2-t1
--FP production rate = dP/dt
Open matlab software and run "growth_rate_module.m" file script. (Instructions for file naming etc can be found in the script)