Difference between revisions of "Team:UC San Diego/Interlab"

Devices Used

GFP generator I13504 was expressed under promoters J23101, J23106, and J23117 to form devices 1, 2, and 3, respectively. GFP device I20270 was used as a positive control and promoter R0040 was used as a negative control. DH5-alpha E. coli was used as a chassis organism. All DNA samples were obtained from the Spring 2015 DNA Distribution Kit. All final constructs were contained in backbone pSB1C3.

Assembly

After amplification, the promoter-containing plasmids were digested with SpeI and PstI, while the GFP-containing plasmid was digested with XbaI and PstI. The GFP generator was gel purified and ligated into each of the promoter-containing plasmids with NEB Quick T4 DNA ligase. The completed devices were verified by sequencing.

Growth Conditions

For each sample, 5mL of bacterial culture in selective LB media was cultured in plastic, 14mL test tubes overnight. Antibiotic was added from a 1000x stock kept at -4°C. The tubes were kept at 37°C and shaken at 300 RPM. The shaker used was a New Brunswick Scientific Excella E24, with a shaking diameter of 19mm. Cultures were prepared in triplicate.

Sampling

After measurement of sample OD600, the cells were resuspended in water to a final volume of 500µL and a final OD600 of 0.5 for plate reader measurement. Fluorescence measurements were also taken in the cells’ growth phase. To do this, sample OD600 was measured, then cells were resuspended in LB media for a final volume of 3mL and final OD600 of 0.1. These cells were allowed to grow for five hours before resuspension in water to a final volume of 500µL and a final OD600 of 0.5 for plate reader measurement.

Measurement

Three 150µL technical replicates from each 500µL sample were loaded into a 96-well plate. Fluorescence measurements were performed on a Tecan Infinite M200 Pro plate reader. Measurement parameters are recorded in the following table:

Results

Data is reported in arbitrary fluorescence units normalized by OD600, giving relative fluorescence in terms of cellular density.