Team:Stony Brook/InterLab
InterLab
This year, our team participated in the iGEM Fourth InterLab study. The objective of this study is to improve the measurement tools in order to obtain more reliable data within the field of synthetic biology. This year’s study aimed to improve the GFP and OD600 measurement protocol via the use of a plate reader. We were provided with 8 plasmids, including 2 controls and 6 test devices, to transform into DH5-alpha Escherichia Coli cells. We measured the fluorescence and absorbance with the Molecular Devices Microplate Reader SoftMax Pro 6.5.1.
Part One
The initial procedure we carried out was to measure the OD600 reference point of LUDOX-S40. The absorbance readings are listed in the table below.
Part Two
The second part of the study was to construct a standard curve by plotting the fluorescence of fluorescin at different concentrations. The table below shows the values collected from the readings and the standard curve generated from the data. As expected, there is a positive correlation between the concentration of fluorescin in uM and the intensity of the fluorescence.
Part Three
The third part of the study is transform our 8 plasmids into DH5-alpha E. coli cells on chloramphenicol plates. We made separate liquid cultures of for two separate colonies per plate. After taking the OD600 of the overnight cultures, we diluted the cells in LB and chloramphenicol according to the volumes below in order to obtain a target absorbance of 0.02.
Colony 1
Colony 2
Then we took the fluorescence and absorbance readings on our plate reader for the time intervals, 0 hours, 2 hours, 4 hours, and 6 hours. The raw readings are listed in the tables below.
If we compare the absorbance readings for Colony 1 and Relicate 1 for all transformations over the course of the 2-hour increments, we see that there is a general increase in the absorbance of cells for most of the plates. As expected, we can infer the cells are growing over time, thus absorbing more light. We also notice that there are low levels of absorbance in Test Device 1 compared to the other devices and controls.
Illustrated here is the raw fluorescence readings for Colony 1 and Relicate 1 for all transformations over the course of the experiment. We see that there is a general increase in the fluorescence of cells for most of the plates. As expected, there is low fluorescence levels in our negative control across all time points throughout the experiment. Test Device 3 also displayed low fluorescence,thus had low GFP productions in the E. coli.
