Team:NJU-China/InterLab

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Introduction

Reliable and repeatable measurement is a key component to all engineering disciplines. We, NJU-China team, participated in this year's InterLab work and uploaded the data and relative questionnaire. In this page, we will show our results, list materials and equipment used in the InterLab Study, and explain the details of our method.

Results

Calibration

In order to take measurements under the consistent conditions, we performed the calibration measurements before the Cell measurements.

OD₆₀₀ reference point

First, we use LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform the absorbance (Abs₆₀₀) data from plate reader into a comparable OD₆₀₀ measurement as would be obtained in a spectrophotometer.

	LUDOX CL-X	H2O
Replicate 1	0.118	0.096
Replicate 2	0.117	0.093
Replicate 3	0.115	0.093
Replicate 4	0.117	0.094
Arith. Mean	0.117	0.094
Corrected Abs600	0.023
Reference OD600	0.063
OD600/Abs600	2.769

Particle standard curve

Second, to convert Abs₆₀₀ measurements to an estimated number of cells, we build a dilution series of monodisperse silica microspheres and measure their Abs₆₀₀ reads in the plate reader to construct a standard curve of particle concentration.

Fluorescein standard curve

Third, we generate a standard curve of fluorescence for fluorescein concentration. This is used to convert our cell-based readings to an equivalent fluorescein concentration.

Cell measurements

We successfully transform Escherichia coli DH5α with given plasmids. We did the measurement after transformation using exactly the same settings with Calibration Procedure. The fluorescence and absorbance readings are shown in Supplemental information.

CFUs per 0.1 OD₆₀₀ E. coli cultures

In order to calibrate OD₆₀₀ to colony forming unit counts, we dilute each culture to 0.1 OD₆₀₀. Then make a serial dilution with each culture and spread plates for these dilutions aseptically. Incubate at 37°C overnight and count colonies on each plate.The results are shown below.

Concentration	10^-3	10^-4	10^-5	OD Value
P-1-1	TMTC	68	12	0.095
P-1-2	TMTC	79	6	0.096
P-1-3	TMTC	50	5	0.099
P-2-1	TMTC	60	4	0.106
P-2-2	TMTC	69	7	0.107
P-2-3	TMTC	57	3	0.109
N-1-1	TMTC	72	8	0.097
N-1-2	TMTC	50	6	0.109
N-1-3	TMTC	54	5	0.108
N-2-1	TMTC	65	8	0.103
N-2-2	TMTC	43	7	0.103
N-2-3	TMTC	75	13	0.106

Materials and Methods

Transformation

10 μL ddH₂O was used to resuspend DNA Distribution Kit wells. Pipet up and down several times and let sit for a few minutes. The solution turned red due to the cresol red dye. 2mL tubes were placed in floating tube racks for better support when working on ice and the heat shock. 2mL tubes and the competent cells were pre-chilled with ice in the bucket. 50μL competent cells were pipetted into 2mL tubes, together with 1μL DNA. Tubes were incubated on ice for 30 min, followed by a heat shock at 42℃ for 1 min. The tubes were incubated on ice for 5 min and added 200μL LB media. The transformations were incubated at 37℃ for 2 hours on a shaker. Each transform was added on two Petri plates for a 20μL and 200μL plating. Overnight (14-18 hours )incubation of transformations at 37℃. Single colonies were picked and counted for control transformation.

Calibration

We add 100 µl LUDOX- S40 (provided in the kit) and 100 µl of H₂O into 96 well plates with flat and transparent bottoms. Absorbance 600 nm of all samples in all standard measurement modes were measured in the instrument whose path length correction was turned off (Spectramax M2). This instrument measured all cell density readings with the same settings and volume. A serial dilution by consecutively transferring 100 µL from column to column with good mixing was performed. Fluorescence of all samples was measured in all standard measurement modes in the instrument (Spectramax M2).

Cell measurements

Escherichia coli DH5α was transformed with these following plasmids.

Positive control	BBa_R0040
Negative control	BBa_I20270
Test Device 1	BBa_J364000
Test Device 2	BBa_J364001
Test Device 3	BBa_J364002
Test Device 4	BBa_J364007
Test Device 5	BBa_J364008
Test Device 6	BBa_J364009

2 colonies from each of plate were picked and inoculated on 5-10 mL LB medium + Chloramphenicol. The cells were grown overnight (16-18 hours) at 37°C and 220 rpm. The instrument was set to read OD₆₀₀ (as OD calibration setting). OD₆₀₀ of the overnight cultures was measured and recorded data. The cultures were diluted to a target OD₆₀₀ of 0.02 in 12 ml LB medium + Chloramphenicol in 15 mL tubes covered with foil to block light. The cultures were incubated at 37°C and 220 rpm. 500 µL samples of the cultures at 0 and 6 hours of incubation were taken. (At each time point, a sample from each of the 8 devices, two colonies per device, for a total of 16 samples will be taken). Samples were placed on ice. Samples were measured (OD and Fl measurement) at the end of the sampling point.

Acknowledgements

This study was supported by M3 Laboratory, School of Life Sciences, Nanjing University. We thank Prof. Qipeng Zhang and Prof. Chen-yu Zhang for their supervision.

Supplemental Information

Supplemental information is in a excel table, which can be found and downloaded here.