iGEM 2018 InterLab Study

Introduction to the InterLab Study

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. GFP was chosen 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.

The aim is 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 as an 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 cell count in the different teams samples, two orthogonal approaches were be used:

1. Converting between absorbance of cells to absorbance of a known concentration of 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.

Overview, Team, and Biosafety Information

EPFL iGEM 2018 Contact:

• Moustafa Houmani (moustafa.houmani@epfl.ch)
• Daniel Nakhaee-Zadeh Gutierrez (daniel.nakhaee-zadehgutierrez@epfl.ch)

People involved in the Interlab Study:

• Moustafa Houmani: Performing calibration protocols; E. coli cell transformation, culturing, growth, sampling, and assay; CFU protocol Sample Preparation, CFU protocol series dilution and plating, CFU protocol cell counting
• Violetta Zanotti: Performing calibration protocols
• Reza Hosseini: E. coli Cell Transformation, culturing, and growth
• Daniel Zadeh: CFU protocol series dilution and plating

We would like to thank our advisor Michael Andrew Crone for his continuous help and support during the Interlab Measurement Study.

Chassis used for the Interlab Measurement Study:

E. coli K-12 DH-5-alpha

Personal Protective Equipment utilized during the experiments:

Lab coats, gloves, long pants

Calibration Measurements

Brand and Model of the plate reader:

PerkinElmer VICTOR X3 multimode plate reader

Measurements Taken:

Both Fluorescence and Absorbance using Bottom Optics

Types of Plates Used:

Black plates with transparent flat bottom

Temperature Setting Used during measurements:

Room Temperature

Calibration Protocols

CALIBRATION PROTOCOLS WERE COMPLETED BEFORE CELL MEASUREMENTS WERE TAKEN!

Three sets of unit calibration measurements were taken: an OD 600 reference point, a particle standard curve, and a fluorescein standard curve. For all of these calibration measurements, we used the same plates and volumes that we later used in the cell-based assays. We also used the same settings (e.g., filters or excitation and emission wavelengths) that we later used in our cell-based assays.

Calibration 1: OD6 00 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 MUST THEREFORE TURN OFF PATHLENGTH CORRECTION if it can be disabled on your instrument . Our Instrument did not have any pathlength correction].

Filter Used for Absorbance Measurements:

595 nm

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)

Method

• Add 100 μl LUDOX into wells A1, B1, C1, D1
• Add 100μl of ddH2 O into wells A2,B2,C2,D2
• Measure absorbance at 600 nm 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

	LUDOX CL-X	H20
Replicate 1	0.046	0.026
Replicate 2	0.045	0.026
Replicate 3	0.045	0.026
Replicate 4	0.047	0.027
Arith. Mean	0.046	0.026
Corrected Abs600	0.020	NA
Reference OD600	0.063	NA
OD600/Abs600	3.231	NA

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 3.231.

Calibration 2: Particle Standard Curve - Microsphere Protocol

We prepared a dilution series of monodisperse 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 beadsMicrosphere suspension (provided in kit, 4.7*10⁸ microspheres)
• ddH₂O (provided by EPFL)
• 96 well plates, black with clear flat bottom (provided by team)

Absorbance Filter Used:

595 nm

Silica Microsphere Properties:

Cospheric Monodisperse Silica Microspheres 0.961 &mu m diameter
Spheres per mass	[- / g]	1.20*10<sup>12</sup>
Density	[g / mL]	1.8
Spheres / 0.55	[- / mL]	1.19*10<sup>12</sup>
Resuspend Volume	[mL]	2.55
Dilution	[X]	100
Total volume	[mL]	255
Particle density	[- / mL]	4.71*10<sup>9</sup>
Well Volume	[mL]	0.1
Initial particles	[-]	4.71*10<sup>8</sup>

Method:

Preparation of the Microsphere stock solution:

• Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex 4 vigorously for 30 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 pipet 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.