This year,our team had been invited to collaborate in the InterLab study, aimed to comparing the variability of measurements among different research groups.
This year, labs should test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable.We don’t know How reliable will these devices turn out to be in labs around the world,so this work will help us make this a more robust engineering standard for measuring GFP in synthetic biology.
For this experiment, we followed the protocol available in the iGEM website.
Prior to starting the measurement protocols below, we transform the 8 plasmids (Positive Control, Negative Control, Test Device 1, Test Device 2, Test Device 3, Test Device 4, Test Device 5, and Test Device 6 - locations listed below) from Kit Plate 6 into E. coli DH5-alpha cells.,accoding to the Transformation Protocol.
We cultured these transformed cells on LB plates with the addition of chloramphenicol. The transformed cells were incubated in this manner overnight for more than 12h at 37ºC and 220rpm,and picked five clonies from each plate. The presence of the correct device in the colonies was checked first by colony PCR and, if the size of the DNA fragment corresponded to the expected size, were then sent for sequencing. The colonies containing the correct plasmid were then incubated overnight for 16 h at 37ºC in 12 mL conical flasks in 5 mL LB medium with chloramphenicol or streptomycin, respectively.
plate reader protocol
Follow the protocol provided by Interlab study,frst we should use LUDOX-S40 as a single point reference to obtain a ratiometric conversion factor to transform our absorbance data into a standard OD600 measurement.The method is :Add 100 µl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette) and Add 100 µl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette) Measure absorbance 600 nm of all samples in all standard measurement modes in instrument Record the data in notebook Import data into Excel (OD600 reference point tab) Sheet_1 provided by Interlab Study.
Then we should prepare for the fluorescein fluorescence standard curve.Follow protocol,we should Prepare 2x fluorescein stock solution (100µM) by resuspending fluorescein in 1 mL of 1xPBS,and add them to the wells follow the picture2.
Next,we read the plate in our plate reader.with the recommended filters: Excitation 485nm, Emission 530/30,and import the date to the Excel (cell measurement tab) Sheet_1 provided
Then we culture our cells and dilute them in Lysogenic Broth (LB) to OD600 of 0.02 in a 96-well plate.follow the protocol like picture 3.
OD600 Reference point
fluorescein fluorescence standard curve