Model

Interlab Study

Note

Description: the goal and main contents were quoted from iGEM International InterLab Measurement Study

Methods: the protocol was provided by iGEM InterLab Committee and described briefly in here

Results: the experiment and data presented here were all made by members of team Mingdao

Reference: Fifth International InterLab Measurement Study@iGEM

Instrument

The machine in the Biolab of Mingdao High School: Synergy H1 Hybrid Multi-Mode Microplate Reader

Introduction

"Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology. However, the ability to repeat measurements in different labs has been difficult. The Measurement Committee, through the InterLab study, has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. We chose GFP as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein."

Goal for the Fifth InterLab

"The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. Until we reach this point, synthetic biology will not be able to achieve its full potential as an engineering discipline, as labs will not be able to reliably build upon others’ work."

"This year, teams participating in the interlab study helped iGEM to answer the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?"

Calibration Reference

Calibration 1: OD600 Reference point - LUDOX Protocol

Materials

1ml LUDOX CL-X

ddH2O

96 well Black Clear Bottom Plate

Method

↓ Add 100 μl LUDOX into wells A1, B1, C1, D1

↓ Add 100 μl of ddH2O into wells A2,B2,C2,D2

↓ Measure absorbance at 600 nm

↓ Record the data

Result

The table shows the OD600 measured by a spectrophotometer (see table above) and plate reader data for H2O and LUDOX corresponding to the expected results. The corrected Abs600 is calculated by subtracting the mean H2O reading. The reference OD600 is defined as that measured by the reference spectrophotometer. The correction factor to convert measured Abs600 to OD600 is thus the reference OD600 divided by Abs600. All cell density readings using this instrument with the same settings and volume can be converted to OD600 by multiplying by 4.200.

Calibration 2: Particle Standard Curve - Microsphere Protocol

Materials

300 μL silica beads Microsphere suspension

ddH2O

96 well Black Clear Bottom Plates

Method

↓ Obtain Silica Beads in the InterLab Kit

↓ Pipet 96 μL beads into an eppendorf

↓ Add 904 μL of ddH2O to the microspheres

↓ Vortex well to obtain stock Microsphere Solution.

↓ Preparation of microsphere serial dilutions as follows

↓ Measure Abs 600

↓ Record the data

Result

Raw Data

Particle Standard Curve

Particle Standard Curve(log scale)

Calibration 3: Fluorescence standard curve - Fluorescein Protocol

Materials

Fluorescein

10ml 1xPBS

96 well Black Clear Bottom Plate

Method

Prepare the fluorescein stock solution

↓ Spin down fluorescein tube

↓ Add 1 mL to make 10x fluorescein stock solution (100 μM) of 1xPBS.

↓ Dilute 100 μL of 10x fluorescein stock into 900 μL 1xPBS

↓ Prepare the serial dilutions of fluorescein as follows

↓ Measure fluorescence at Ex/Em = 485/528 nm

Result

Raw Data

Fluorescein Standard Curves

Fluorescein Standard Curves(log scale)

Cell Measurement

Prior to performing the cell measurements all three of the calibration measurements should be performed.

For the sake of consistency and reproducibility, Interlab Measurement requires all teams to use E. coli K-12 DH5-alpha.

For all of these cell measurements,we used the same plates and volumes that we used in the calibration protocol.We also used the same settings (e.g., filters or excitation and emission wavelengths) that you used in your calibration measurements.

Materials

 Competent cells ( Escherichia coli strain DH5 )

 LB (Luria Bertani) media

 Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)

 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)

 Incubator at 37°C

 1.5 ml eppendorf tubes for sample storage

 Ice bucket with ice

 Micropipettes and tips

 96 well Black Clear Bottom Plate

Workflow

Method

Day1

transform Escherichia coli DH5 with these following plasmids (all in pSB1C3):

Thermo-Fisher DH5-alpha Competent Cells (Catalogue #: 18265017 were purchased).

iGEM protocols for resuspending DNA from the kit plates and performing the transformation were used:http://parts.igem.org/Help:Protocols/Transformation

Day2

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.

Day 3

Cell growth, sampling, and assay

Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)

Measure Abs 600 of these 1:10 diluted cultures

Record the data in your notebook

Dilute the cultures further to a target Abs6 00 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light)

Take 500 L samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 μl samples per time point, 32 samples total). Place the samples on ice.

Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.

Take 500 μl samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice.

At the end of sampling point you need to measure your samples (Abs600 and fluorescence measurement), see the below for details.

Record data in your notebook

Import data into Excel sheet provided ( fluorescence measurement tab )

Measurement:

Samples should be laid out according to the plate diagram below. Pipette 100 μl of each sample into each well. From 500 μl samples in a 1.5 ml eppendorf tube, 4 replicate samples of colony #1 should be pipetted into wells in rows A, B, C and D. Replicate samples of colony #2 should be pipetted into wells in rows E, F, G and H. Be sure to include 8 control wells containing 100uL each of only LB+chloramphenicol on each plate in column 9, as shown in the diagram below. Set the instrument settings as those that gave the best results in your calibration curves (no measurements off scale). If necessary you can test more than one of the previously calibrated settings to get the best data (no measurements off scale). Instrument temperature should be set to room temperature (approximately 20-25°C) if your instrument has variable temperature settings.

Layout for Abs 600 and fluorescence measurement: