Team:UC San Diego/Interlab

Interlab

The Interlab Measurement Study was conducted by Walter Thavarajah, Vivienne Gunadhi, and Jenny Lee on 7/18/15 - 7/31/15, with support and troubleshooting advice from Phillip Kyriakakis.

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 -4C. The tubes were kept at 37C 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 500uL and a final OD600 of .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 .1. These cells were allowed to grow for five hours before resuspension in water to a final volume of 500uL and a final OD600 of .5 for plate reader measurement.

Measurement

Three 150uL technical replicates from each 500uL 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:

Measurement Parameters

Parameter Value
Mode Fluorescence Top Reading
Shaking Duration 10s
Shaking Amplitude 1mm
Excitation Wavelength 485nm
Emission Wavelength 535nm
Excitation Bandwidth 9nm
Emission Bandwidth 20nm
Gain 109
Number of Flashes 25
Integration Time 20us
Lag Time 0us
Settle Time 0ms
Z-Position (Manual) 15000um

Results

Fluorescence data was normalized by dividing by OD600, giving fluorescence in terms of cellular density. For all devices, the cells that were measured during lag phase showed less fluorescence and higher variability between biological replicates than those measured during the growth phase did. However, both sets of measurements showed similar overall trends. In both groups, Device 1, J23101 + I13504, had the highest emission. Similarly, Devices 2, J23106 + I13504, and 3, J23117 + I13504, showed similar fluorescence in both groups.

Normalized Stationary Phase Fluorescence

Replicate (Bio) 1 2 3
Replicate (Tech) 1 2 3 1 2 3 1 2 3
J23101+I13504 212661 218069 224560 87324 83817 85638 99780 104666 96564
J23106+I13504 91142 90870 91374 138854 135266 138449 78490 76435 76820
J23117+I13504 36714 35889 35927 86677 87505 88218 100962 98886 101123
I20270 59086 58353 58899 66048 66005 65974 23207 23189 22479
R0040 7841 8595 8823 9514 9610 9661 8611 8411 11601

Normalized Growth Phase Fluorescence

Replicate (Bio) 1 2 3
Replicate (Tech) 1 2 3 1 2 3 1 2 3
J23101+I13504 228630 220402 215458 250390 260929 251502 223796 244465 239753
J23106+I13504 137338 136645 138662 152553 152022 151875 155750 154273 156722
J23117+I13504 164102 166495 164769 155177 154736 157905 119813 120808 119416
I20270 132783 133059 135514 111394 113750 117485 138890 126284 128169
R0040 18327 17437 18190 18767 17509 17658 16599 16547 15929

Discussion

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