Team:UCLouvain/InterLab

INTERLAB

Overview

This is the third edition of iGEM for Université catholique de Louvain team and for the second time in a row we decided to take part in the InterLaboratory Measurement Study in synthetic biology. The InterLab is an essential part of the iGEM competition and is an interesting challenge for our team. We estimate that this experience will allow us to increase our awareness about scientific reproducibility. The reproducibility allows to determine the trustworthiness of the results of the same experience carried out in different conditions (different laboratories, equipment, operators). Therefore, iGEM is the perfect opportunity to test the reproducibility of a protocol. Since we are more than 300 teams from all over the world, we namely represent this concept of “different conditions” in terms of places, equipment and operators.

Moreover, we used this experience to “train” the members of our team which had little experience of laboratory work. Even if they were well supervised, this resulted in some manipulation mistakes. Thanks to this experience we became more familiar with the laboratory activities before focusing completely on our own project. In spite of not being directly related to our project, the InterLab allowed us to further develop our skills and to acquire a precise and reliable working

The Team

Those are the team members which took care of the interlab. As you can see, we could all be top-models. Fun fact: Antoine (in the middle) cut his hair for the occasion.

All three boys (Luca -left-, Antoine -you already know him- and Mathieu -right-) took care of the cell measurement protocol. Meanwhile, Nina (left) and Fiona (right) took care of all the rest.

Materials and Settings

OD600 reference point - LUDOX protocol

Materials:

1 ml LUDOX CL-X (provided in kit by iGEM)
ddH2O
96 well plate, black with clear flat bottom
Tecan infinite M200 Pro (plate reader)
micropipette: p100

Settings:

Wavelength: 600 nm
Bandwidth: 9 nm
Number of flashes: 25

Particle standard curve - Microsphere protocol

Materials:

300 μL Silica beads - Microsphere suspension (provided in kit by iGEM, 4.7 x 10^8
ddH2O
96 well plate, black with clear flat bottom
Tecan infinite M200 Pro (plate reader)
vortex - VWR VV3
micropipette: p20, p100, p200, p1000
1,5 ml Eppendorf

Settings:

Wavelength: 600 nm
Bandwidth: 9 nm
Number of flashes: 25

Fluorescence standard curve - Fluorescein protocol

Materials:

Fluorescein (provided in kit by iGEM)
10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline)
96 well plate, black with clear flat bottom
Tecan infinite M200 Pro (plate reader)
centrifuge: 3500 rpm - Eppendorf centrifuge 5424
micropipette: p100, p200, p1000
1,5 ml Eppendorf

Settings:

Excitation wavelength: 485 nm
Emission wavelength: 525 nm
Excitation bandwidth: 9 nm
Emission bandwidth: 20 nm
Gain: 100 manual
Number of flashes: 25
Integration time: 20 μs

Cell measurement protocol

Materials:

Competent cells (Escherichia coli K-12 strain DH5α)
LB (Luria Bertani) media
Petri dishes
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
50 ml Falcon tube covered in foil to block light
Incubator at 37°C
1.5 ml Eppendorf tubes for sample storage
Ice bucket with ice
Micropipettes: p2, p100, p1000
96 well plate, black with clear flat bottom
Electroporation machine - Bio-RAD Gene Pulser XCell^TM
2 mm cuvettes
Pasteur pipettes
Tecan infinite M200 Pro (plate reader)
Bunsen burner
Devices (from Distribution Kit given by iGEM, all in pSB1C3 backbone).

Settings:

Fluorescence:

Excitation wavelength: 485 nm
Emission wavelength: 525 nm
Excitation bandwidth: 9 nm
Emission bandwidth: 20 nm
Gain: 100 manual
Number of flashes: 25
Integration time: 20 μs

Absorbance:

Wavelength: 600 nm
Wavelength: 600 nm
Bandwidth: 9 nm
Number of flashes: 25

Electroporation:

2500 V

Colony Forming Units per 0.1 OD600 E.coli cultures protocol

Materials:

Two positive control (BBa_I20270)
Two negative control (BBa_R0040)
LB (Luria Bertani) media
36 Petri dishes
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
Incubator at 37°C
1.5 ml and 2 ml Eppendorf tubes for sample storage
Micropipettes: p20, p100, p200, p1000
96 well plate, black with clear flat bottom
Tecan infinite M200 Pro (plate reader)
vortex - VWR VV3
Bunsen burner

Methods

The following protocols have been provided by iGEM. They can also be found in PDF version on their website.

OD600 reference point - LUDOX protocol

We used LUDOX CL-X (45% colloidal silica suspension) 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) intocomparable OD600 measurements. The LUDOX solution is only weakly scattering and so will give a low absorbance value.
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. We don't have the pathlength correction on our instrument. 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.

Protocol:

Add 100 μl LUDOX into wells A1, B1, C1, D1
Add 100 μl of dd H2 O into wells A2, B2, C2, D2
Measure absorbance at 600 nm of all samples with the same measurement mode

Particle standard curve - Microsphere protocol

We will prepare a dilution series of monodisperse silica microspheres and measure 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 will allow us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.

Protocol:

Prepare the Microsphere Stock Solution:

Obtain the tube labeled "Silica Beads" from the InterLab test kit and vortex vigorously for 30 seconds. (Note: Microspheres should NOT be stored at 0°C or below, as freezing affects the properties of the microspheres).
Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
Add 904 μL of ddH2 O to the microspheres
Vortex well. This is the Microsphere Stock Solution.

Prepare the serial dilution of Microspheres:

Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. Column 12 must contain ddH2O only.
Initially we will setup the plate with the microsphere stock solution in column 1 and an equal volume of 1 x ddH2O in columns 2 to 12. We will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing.

Add 100 μl of ddH2O into wells A2, B2, C2, D2....A12, B12, C12, D12
Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds
Immediately add 200 μl of microspheres stock solution into A1
Transfer 100 μl of microsphere stock solution from A1 into A2
Mix A2 by pipetting up and down 3x and transfer 100 μl into A3
Mix A3 by pipetting up and down 3x and transfer 100 μl into A4
Mix A4 by pipetting up and down 3x and transfer 100 μl into A5
Mix A5 by pipetting up and down 3x and transfer 100 μl into A6
Mix A6 by pipetting up and down 3x and transfer 100 μl into A7
Mix A7 by pipetting up and down 3x and transfer 100 μl into A8
Mix A8 by pipetting up and down 3x and transfer 100 μl into A9
Mix A9 by pipetting up and down 3x and transfer 100 μl into A10
Mix A10 by pipetting up and down 3x and transfer 100 μl into A11
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!
Repeat dilution series for rows B, C, D
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.
Measure Abs600 of all samples in instrument

Fluorescence standard curve - Fluorescein protocol

Plate readers report fluorescence values in arbitrary units that vary widely from instrument to instrument. Therefore absolute fluorescence values cannot be directly compared from one instrument to another. In order to compare fluorescence output of test devices between teams, it is necessary for each team to create a standard fluorescence curve. Although distribution of a known concentration of GFP protein would be an ideal way to standardize the amount of GFP fluorescence in our E. coli cells, the stability of the protein and the high cost of its purification are problematic. We therefore use the small molecule fluorescein, which has similar excitation and emission properties to GFP, but is cost-effective and easy to prepare. (The version of GFP used in the devices, GFP mut3b, has an excitation maximum at 501 nm and an emission maximum at 511 nm; fluorescein has an excitation maximum at 494 nm and an emission maximum at 525nm).

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 will generate a standard curve of fluorescence for fluorescein concentration. We will be able to use this to convert our cell based readings to an equivalent fluorescein concentration.

Protocol:

Prepare the fluorescein stock solution from dried fluorescein:

Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
Prepare fluorescein stock solution (100 μM - 10x) by resuspending fluorescein in 1 mL of PBS (1x). Note: it is important that the fluorescein is properly dissolved. To check this, after the resuspension you should pipette up and down and examine the solution in the pipette tip - if any particulates are visible in the pipette tip continue to mix the solution until they disappear.
Dilute the fluorescein stock solution (10x) with PBS (1x) to make a fluorescein solution (1x) with concentration 10 μM: 100 μL of fluorescein stock (10x) into 900 μL PBS (1x).

Prepare the serial dilutions of fluorescein:

Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 must contain PBS buffer only.
Initially we will setup the plate with the fluorescein stock in column 1 and an equal volume of PBS (1x) in columns 2 to 12. We will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing.

Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1
Transfer 100 μl of fluorescein stock solution from A1 into A2
Mix A2 by pipetting up and down 3x and transfer 100 μl into A3
Mix A3 by pipetting up and down 3x and transfer 100 μl into A4
Mix A4 by pipetting up and down 3x and transfer 100 μl into A5
Mix A5 by pipetting up and down 3x and transfer 100 μl into A6
Mix A6 by pipetting up and down 3x and transfer 100 μl into A7
Mix A7 by pipetting up and down 3x and transfer 100 μl into A8
Mix A8 by pipetting up and down 3x and transfer 100 μl into A9
Mix A9 by pipetting up and down 3x and transfer 100 μl into A10
Mix A10 by pipetting up and down 3x and transfer 100 μl into A11
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!
Repeat dilution series for rows B, C, D
Measure Abs600 of all samples in instrument

Electroporation protocol

Please refer to this protocol.

Cell measurement protocol

Prior to performing the cell measurements we performed all three of the calibration measurements. For the take of consistency and reproducibility, we used E. coli K-12 DH5-alpha, as the other teams. 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 that in our calibration measurements.

Protocol:

Day 1:Transform Escherichia coli DH5α with the plasmids (all in pSB1C3) given in the material and settings section. Please refer to the electroporation protocol.
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.
Day3: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 Abs600 of these 1:10 diluted cultures
Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12ml LB medium + Chloramphenicol in 50 mL falcon tube (covered with foil to block light).
Take 500 μL 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 we need to measure your samples (Abs600 and fluorescence measurement), see the below for details.

Measurement:

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. The setting used were the same as those used for the calibration protocols.

Layout for Abs600 and Fluorescence measurement:

At the end of the experiment, we have two plates to read. We have one plate for each time point: 0 and 6 hours. On each plate we will read both fluorescence and absorbance.

Colony Forming Units per 0.1 OD600 E.coli cultures protocol

This procedure can be used to calibrate OD600 to colony forming unit (CFU) counts, which are directly relatable to the cell concentration of the culture, i.e. viable cell counts per mL. This protocol assumes that one bacterial cell will give rise to one colony.

For the CFU protocol, we will count colonies for our two Positive Control (BBa_I20270) cultures and our two Negative Control (BBa_R0040) cultures.

Step 1: Starting Sample Preparation
This protocol will result in CFU/mL for 0.1 OD600. Our overnight cultures will have a much higher OD600 and so this section of the protocol, called "Starting Sample Preparation", will give us the "Starting Sample" with a 0.1 OD600 measurement.

Measure the OD600 of the cell cultures, making sure to dilute to the linear detection range of the plate reader. In our case, the range is between 0.097 and 0.239. Include blank media (LB + Cam) as well.
For an overnight culture (16-18 hours of growth), we recommend diluting the cultures 1:8 (8-fold dilution) in LB + Cam before measuring the OD600.

Preparation: Add 25 μL culture to 175 μL LB + Cam in a well in a black 96-well plate, with a clear, flat bottom. Recommended plate setup is below. Each well should have 200 μL.
The scheme is the following:
- A1 - A2: Positive Controls (cultures 1-2)
- B1 - B2: Negative Controls (cultures 3-4)
- C1 - C2: Blank media - 200 μL of LB + Cam (in duplicate)

Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cam media. Do this in triplicate for each culture.
Use (C1)(V1) = (C2)(V2) to calculate the dilutions, where:
- C1 is your starting OD600 C2 is the target OD600 of 0.1
- V1 is the unknown volume in μL
- V2 is the final volume of 1000 μL
Important: When calculating C1, subtract the blank from the reading and multiple by the dilution factor used.
Check the OD600 and make sure it is 0.1 (minus the blank measurement). The scheme is the following:

A1 - A2: Positive Controls (cultures 1-2)
B1 - B2: Negative Controls (cultures 3-4)
C1 - C2: Blank media - 200 μL of LB + Cam (in duplicate)
A3 - A8: 0.1 Starting Sample Dilutions for Positive Controls (in triplicate per culture, 6 total dilutions)
B3 - B8: 0.1 Starting Samples Dilutions for Negative Controls (in triplicate per culture, 6 total dilutions)

Step 2: Dilution Series Instructions
We made the following serial dilutions for the triplicate Starting Samples you prepared in Step 1. We should have 12 total Starting Samples - 6 for your Positive Controls and 6 for your Negative Controls.
For each Starting Sample :

3 LB Agar + Cam plates (36 total) is needed.
Prepare three 2.0 mL tubes (36 total) with 1900 μL of LB + Cam media for Dilutions 1, 2, and 3.
Prepare two 1.5 mL tubes (24 total) with 900 μL of LB + Cam media for Dilutions 4 and 5.
Label each tube for each Starting Sample.
Pipet 100 μL of Starting Culture into Dilution 1. Discard tip. Do not pipette up and down.
Vortex tube for 5-10 secs.
Repeat Step 5 for each dilution through to Dilution 5.
Aseptically spead plate 100 μL on LB + Cam plates for Dilutions 3, 4, and 5.
Incubate at 37°C overnight and count colonies after 18-20 hours of growth.

Step 3: CFU/mL/OD Calculation Instructions
Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture can be calculated as follows:

Count the colonies on each plate with fewer than 300 colonies.
Multiple the colony count by the Final Dilution Factor on each plate.

Results and Discussion

OD600 reference point

The four replicates of LUDOX and ddH2O give reproducible results. We therefore obtain OD600/Abs600=3,679.

Particle Standard Curve

To obtain better results, we did not take the most concentrated point into account. On both linear and log scale, we observe that the values form a straight line. Discrepancies between the replicates can be explained by the fact that the light beam of the Tecan machine does not always pass by the same point of each well. Beside, we observed water vapor on the well plate cap, this probably altered a bit the volume in the wells.

Fluorescence standard curve

We observe that the values form a straight line on both linear and log scale. Beside, we observed water vapor on the well plate cap, this probably altered a bit the volume in the wells.

Cell measurement

We can see that both absorbance600 and fluorescence values rise.
The factors used to pass from the absorbance and the fluorescence values to fluorescence per OD and fluorescence per particles values are the following:
1. OD600 / Abs600 = 3.68
2. uM Fluorescein / a.u. = 1.59E-03
3. Particles / Abs600 = 7.25E+08
4. MEFL / a.u. =9.55E+09

Some values of fluorescence where too high to be computed by our machine. For those, we used the maximal value of the machine in the computations. Corresponding values are indicated by red boxes.

Colony Forming Units per 0.1 OD600 E.coli cultures

Raw results

As expected, the number of colonies falls with the dilutions. We chose to use the result of the dilution 4(8x10^5) to compute the concentration of CFU/ml.
Our results could be a bit distorted by the fact that there was water on the petri dishes we used as they did not dried under a hood.
The first Petri dish (on the left) was used to control potential contaminations. Nothing has grown on this plate. The left (resp. right) block of 18 plates corresponds to the culture of different dilutions of positive (resp. negative) controls: The first column represents dilution 3, the next corresponds to dilution 4 and the last column is dilution 5.

To go Further

To smoothen the functioning of the interlabs, we wish we had access to more theoretical documents to understand in more details the goal of the experiments, as well as the computation of the excel file.