Team:BIT-China/InterLab

InterLab

Introduction

Interlab is aimed to improve reproducibility and compare the difference between laboratories. To complete this job,we make three sets of unit calibration measurements: an OD600 reference point, a particle standard curve, and a fluorescein standard curve. Completion of the calibrations will ensure that we understand the measurement process and that we can take the cell measurements under the same conditions. For the sake of consistency and reproducibility, we used E. coli K-12 DH5-alpha like other teams. Later, we conducted the CFU experiment. We count colonies for our two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures.

Methods

—— OD 600 Reference point - LUDOX Protocol

We use LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform your absorbance (Ab_S600) data from our plate reader into a comparable OD₆₀₀ measurement.

$$ Correction\ factor=\frac{ReferenceOD_{600}}{reading_{LUDOX CL-X}-reading_{H_2 O}} $$

—— Particle Standard Curve - Microsphere Protocol

We prepare a dilution series of monodisperse silica microspheres and measure the Ab_S600 in our plate reader. This measurement allow us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.

$\surd$ Prepare the Microsphere Stock Solution

$\surd$ Prepare the serial dilution of Microspheres

$\surd$ Measure Ab_S600 of all samples in instrument

——Fluorescence standard curve - Fluorescein Protocol

We will prepare a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in our plate reader. By measuring these in our plate reader,we generate a standard curve of fluorescence for fluorescein concentration. Thus we are able to use this to convert our cell based readings to an equivalent fluorescein concentration.

$\surd$ Prepare the fluorescein stock solution

$\surd$ Prepare the serial dilutions of fluorescein

$\surd$ Measure fluorescence of all samples in instrument

—— Cell measurement protocol

For all of these cell measurements, we use the same plates and volumes that we used in our calibration protocol. For the first time,we have measurements that are off scale (“OVERFLOW”), so we adjust our settings to make the data in range. Finally, we get the right data.

$\surd$ Transform Escherichia coli DH5α with these following plasmids

Negative control	BBa_R0040	Kit Plate 7 Well 2D
Positive control	BBa_I20270	Kit Plate 7 Well 2B
Test Device 1	BBa_J364000	Kit Plate 7 Well 2F
Test Device 2	BBa_J364001	Kit Plate 7 Well 2H
Test Device 3	BBa_J364002	Kit Plate 7 Well 2J
Test Device 4	BBa_J364007	Kit Plate 7 Well 2L
Test Device 5	BBa_J364008	Kit Plate 7 Well 2N
Test Device 6	BBa_J364009	Kit Plate 7 Well 2P

$\surd$ Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium +Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm

$\surd$ Cell growth, sampling, and measurement

——Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures

This procedure is used to calibrate OD600 to colony forming unit (CFU) counts, which are directly relatable to the cell concentration of the culture. For the CFU protocol, we count colonies for our two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures.

$\surd$ Starting Sample Preparation

$\surd$ Dilution Series Instructions

$\surd$ Calculation

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