Team:Alverno CA/InterLab
Interlab Study
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.
Protocol:
Results:
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| FITC Fluorescence standard curve. Row A had pipetting errors and so is different from the other curves. |
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| Graph of Absorbance (600) over a 6 hour time period |
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| Graph of Fluorescence in AU over a 6 hour time period |
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| Graph of Fluorescence/Absorbance in AU over a 6 hour time period |
