Quantifying different sources of variation in gene expression using
fluorescent transformed E. coli bacteria
Summary:
As part of the 2016 iGEM Interlab Study, we tested 3 different
fluorescent protein expression plasmids, J23101, J23106, and J23117 with
positive and negative controls. We transformed the parts with iGEM's
transformation protocol. Our bacteria were cultured on a LB agar plate and
cultured in LB broth. To measure the differences in fluorescence expressed
by different strength plasmids, we used a plate reader with an OD600
reference point and FITC standard curve.
Project Description:
The 2016 iGEM Interlab Study aims to compare results obtained by various
teams in order to quantify the expression of 5 different constructs using
fluorescence, which provides a useful insight into expression levels that
can be monitored without disrupting cells. Each device used in the study is
in the pSB1C3 plasmid backbone and are fluorescent protein expression
plasmids of different strength promoters. The devices we tested were
J23101, J23106, J23117 (Test Devices 1, 2, and 3). We used positive control
I20270 and negative control R0040. J23101, with a strong promoter, showed
the highest amount of fluorescence. J23117, with a weak promoter, showed
the lowest amount of fluorescence, and J23106, with the medium strength
promoter, showed fluorescence amounts between the other two. Our test
devices were inserted into DH5alpha E. coli, which were transformed
according to the iGEM transformation protocol. Our transformed cells were
plated on LB plates with chloramphenicol and incubated overnight at 37° C.
Two colonies were picked from each plate (with the exception of Test Device
3, with which there was only one) and were inoculated in 15mL test tubes.
These devices were diluted, and then measured in a plate reader with an
excitation wavelength of 35nm, according to iGEM’s measurement
protocol.
Graph of Absorbance (600) over a 6 hour
time period
Graph of Fluorescence in AU over a 6
hour time period
Graph of Fluorescence/Absorbance in AU
over a 6 hour time period
FITC Fluorescence standard curve. Row A had
pipetting errors and so is different from the other
curves.
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