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 -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

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)	One			Two			Three
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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