InterLab Protocol & Worksheet

Individuals responsible for conducting InterLab study:

Rabea Jesser, Julia Donauer (Plate reader measurements)
Ramona Emig, Lara Stühn, Julika Neumann (cloning)
Julian Bender (Plate reader measurements, data processing)

Date of InterLab Study

2015/08/11, 2015/08/13 & 2015/08/15

What chassis did you use?

E.coli TOP10 E.coli ArcticExpress (DE3)RP

Please provide the positive control(s) you used.

BBa_I20270

Please provide the negative control(s) you used.

Untransformed for both strains Transformed with BBa_R0040 (ptetR in pSB1C3)

What type of agar did you use?

LB Agar with 30µg/mL chloramphenicol

Type of incubator

NewBrunswick Innova R 44 Shaker 2.5 cm orbit

Workflow

Streak out 1 plate per device and control
Incubate plates for 17h at 37 C
Inoculate test tube with dimension (WxH) of 16 x 1.3 cm and 5mL LB-medium 30µg/mL chloramphenicol
Incubate test tubes for 8h in incubator with 220 rpm at 37°C
For the 10°C samples: transfer them to 10°C incubator
For the 37°C samples: shake another 8 h in 37°C incubator

Set your instrument to read OD₆₀₀
Measure each sample in cuvette
Take the measurement and record it
Measure OD₆₀₀ of all samples
Calculate the dilution required for each sample
Dilute each sample
Re-measure your sample on OD₆₀₀
If your OD₆₀₀ is within 5% of 0.5, proceed
If your OD₆₀₀ is outside that range, recalculate your dilution and remeasure until it's within the range of +/- 5%

set up sodium fluoresceine standard curve with 500, 375, 250, 125, 50, 25, 10, 5 and 0 ng/mL concentration

pipette the samples and the standard curve in 96-well plates (triplicates)
measure emission at 530 nm after excitation at 485 nm

Measuring and Evaluation

Measuring device

BioTek Synergy H4 Plate Reader (calibrates itself before each measurement) Excitation: 485 nm / 20nm bandpass Emission: 530 nm / 20 nm bandpass Sampling frequency: End-point kinetics

Differences from the InterLab Protocol

To test the influence of temperature and the strain used on the device expression we performed the protocol four times, with a Top10 or an Arctic E. coli strain and at 10°C and 37°C respectively. Therefore we two times exactly followed the protocol provided and two times - after incubating 8h at 37 °C - changed the incubation temperature to 10°C for another 8h.

Data evaluation

For the technical replicates raw data from three measurements of the same sample were averaged and the standard deviation was computed. From the standard deviations of one condition (10°C or 37°C) the maximum, minimum and average value was determined. The values for the biological replicates (thus the means of the technical replicates) were then averaged. To estimate an error for this value the minimum relative error from the technical replicates was subtracted from the relative standard deviation (comprising of the technical and the biological deviations) using Gaussian error propagation. The remaining error should show the maximum biological variability possible in agreement with our data. To obtain absolute values for the fluorescence the slope and intercept of a sodium fluorescein standard curve was used to convert the relative fluorescence units. These values were multiplied with the relative errors yielded above to get absolute measures for the biological variability in the samples. The controls were not used to compute the final data but as a measure to check if the gained data may be reliable.

info, uploaded

Team:Freiburg/InterLab Study/Protocol