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Interlab Study

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 forth is 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.

The aim to improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole. One of the big challenges in synthetic biology measurement has been that fluorescence data usually cannot be compared because it has been reported in different units or because different groups process data in different ways. Many have tried to work around this using “relative expression” comparisons; however, being unable to directly compare measurements makes it harder to debug engineered biological constructs, harder to effectively share constructs between labs, and harder even to just interpret your experimental controls.

The InterLab protocol aims to address these issues by providing researchers with a detailed protocol and data analysis form that yields absolute units for measurements of GFP in a plate reader.

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 asan engineering discipline, as labs will not be able to reliably build upon others’work.

In the previous interlab studies, it was shown that by measuring GFP expression in absolute fluorescence units calibrated against a known concentration of fluorescent molecule can greatly reduce the variability in measurements between labs.However,when taking bulk measurements of a population of cells (such as with a plate reader), there is still a large source of variability in these measurements: the number of cells in the sample.

Because the fluorescence value measured by a plate reader is an aggregate measurement of an entire population of cells, we need to divide the total fluorescence by the number of cells in order to determine the mean expression level of GFP per cell. Usually this is done by measuring the absorbance of light at 600nm, from which the “optical density (OD)”of the sample is computed as an approximation of the number of cells. OD measurements are subject to high variability between labs, however,and it is unclear how good of an approximation an OD measurement actually is. If a more direct method is used to determine the cell count in each sample, then potentially another source of variability can be removed from the measurements.

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?

In order to compute the cellcount in the different teams samples, two orthogonal approaches were be used:

1. Converting between absorbance of cellsto absorbance of a known concentrationof beads.

Absorbance measurements use the way that a sample of cells in liquid scatter light in order to approximate the concentration of cells in the sample. In this year’s Measurement Kit, teams were provided with a sample containing silica beads that are roughly the same size and shape as a typical E. coli cell, so that it should scatter light in a similar way.Because the concentration of the beads is known, each lab’s absorbance measurements can be converted into a universal, standard “equivalent concentration of beads” measurement.

2. Counting colony-forming units(CFUs) from the sample.

A simple way to determine the number of cells in a sample of liquid media is to pour some out on a plate and see how many colonies grow on the plate. Since each colony begins as a single cell(for cells that do not stick together), we can determine how many live cells were in the volume of media that we plated out and obtain a cell concentration for our sample as a whole.Each team will have to determine the number of CFUs in positive and negative control samples in order to compute a conversion factor from absorbance to CFU. By using these two approaches, Interlab Measurement Study will be able to determine how much they agree with each other,and whether using one(or both)can help to reduce lab-to-lab variability in measurements. If it can, then together we will have brought synthetic biology one step closer to becoming a true, reliable engineering discipline.

Calibration Reference

Calibration 1: OD600 Reference point - LUDOX Protocol

LUDOX CL-X (45%colloidal silica suspension) was used as a single point reference to obtain a conversion factor to transform our absorbance (Abs600) data from our plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer. Such conversion is necessary because plate reader measurements of absorbance are volume dependent; the depth of the fluid in the well defines the path length of the light passing through the sample, which can vary slightly from well to well. In a standard spectrophotometer, the path length is fixed and is defined by the width of the cuvette, which is constant.Therefore this conversion calculation can transform Abs600 measurements from a plate reader (i.e., absorbance at 600nm, the basic output of most instruments) into comparable OD600 measurements.The LUDOX solution is only weakly scattering and so will give a low absorbance value.
[ IMPORTANT NOTE : many plate readers have an automatic path length correction feature. This adjustment compromises the accuracy of measurement in highly light scattering solutions, such as dense cultures of cells.YOU MUSTTHEREFORETURN OFF PATH LENGTH CORRECTION if it can be disabled on your instrument . Our Instrument did not have any path length correction].
Materials
1ml LUDOX CL-X (provided in kit)
ddH2 0 (provided by team)
96 well plate, black with clear flat bottom preferred (provided by team)
Methods
Add 100 μl LUDOX into wells A1, B1, C1, D1
Add 100 μl of ddH2 O into wells A2, B2, C2, D2
Measure absorbance at 600nm of all samples in the measurement mode you plan to use for cell measurements
Record the data in the table below or in your notebook
Import data into Excel sheet provided ( OD600 reference point tab )
Results

The table shows the OD600 measured bya 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
We prepared a dilution series of mono disperse silica microspheres and measured the Abs600 in our plate reader.The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume.This measurement allows us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.
Materials
300 μL silica beads Microsphere suspension (provided in kit, 4.7*108 microspheres)
ddH2O (provided byEPFL)
96 well plates, black with clear flat bottom(provided by team)
Methods
Preparation of the Microsphere stock solution:
Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex 4 vigorously for30 seconds. NOTE: Microspheres should NOT be stored at 0 ° C or below, as freezing affects the properties of the microspheres. If you believe your microspheres may have been frozen, please contact the iGEM Measurement Committee for a replacement (measurement at igem dot org).
Immediately pipette 96 μL eppendorf
Add 904 μL of ddH2O to the microspheres
Vortex well to obtain stock Microsphere Solution.
Preparation of microsphere serial dilutions:
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 MUST CONTAIN ddH2O ONLY.Initially you will setup the plate with the microsphere stock solution in column 1 and an equal volume of 1x ddH2O in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μL from column to column with good mixing.

1. Add 100 μl of ddH2O into wells A2, B2, C2, D2....A12, B12, C12, D12
2. Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds
3. Immediately add 200 μl of microspheres stock solution into A1
4. Transfer 100 μl of microsphere stock solution from A1 into A2.
5. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3
6. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
7. Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
8. Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
9. Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
10. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...
11. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9…
12. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
13. Mix A10 by pipetting up and down 3x and transfer 100μl into A11...
14. Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste
TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.
15. IMPORTANT! Re-Mix (Pipette up and down) each row of your plate immediately before putting in the plate reader! (This is important because the beads begin to settle to the bottom of the wells within about 10 minutes, which will affect the measurements.)Take care to mix gently and avoid creating bubbles on the surface of the liquid.
16. Measure Abs600 of all samples in instrument
17. Record the data in your notebook
18. Import data into Excel sheet provided ( particle standard curve tab )
Results
Raw Data

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