Team:Glasgow/Interlab

Glasglow

Interlab Study

Overview

All 2015 iGEM teams have been invited to participate in the Second International InterLab Measurement Study in synthetic biology. Each lab will obtain fluorescence data for the same three GFP-coding devices with different promoters varying in strength. The objective is to assess the robustness of standard parts and the variability of measurements among different research groups using different lab techniques.

Introduction

This year iGEM Glasgow have participated in the InterLab study and Extra Credit. The three devices required were cloned, as specified, and using a plate reader measurements were obtained in absolute units in terms of moles of FAM labelled oligonucleotide.

Release

Individuals responsible for conducting InterLab study

Charlotte Flynn - Carried out cloning of devices, measurements of devices and completed the relevant forms and content of wiki page.

Others who should be credited, e.g., in a publication based on this data

Vilija Lomeikaite - Set up overnight cultures of the devices
Ye Yang - Designed and formatted the Interlab wiki page
Andrey Filipov - Carried out the calibration measurements for the spectrophotometer
James Provan - Sent cloned devices for sequencing

Dates of InterLab Study

The cloning of devices was carried out from the 17 – 21st of August. Measurements of the devices were carried out from the 24th– 28th of August.

Equipment

Equipment used to acquire measurements

Model and manufacturer:
o Incubator – 2cm shaking diameter
o Spectrometer – Used to measure absorbance at 600nm of each sample.
o Typhoon FLA 9500 - GE Healthcare Life Sciences. Wavelength used to excite cells - 475nm. Filter/channel used to capture the light emission from the cells - Filter BPB1 (530DF20).

Spectrophotometer calibration

In order to calibrate the spectrophotometer a dilution series of 1-100% of DH5 alpha cells was carried out and the A600 of each sample was measured (Figure 1).

Typhoon FLA 9500 calibration

A dilution series was measured for phiLOV protein (Figure 2), converted to numerical readings (Table 1) and calibration curve (Figure 3) carried out to calibrate the Typhoon.

Figure 2: Fluorescence readings of a dilution series of phiLOV. 67.5µg = 67.5µg phiLOV in 100µl PBS. Each concentration was carried out twice.

Table 1: Summary of the fluorescence readings of phiLOV protein.

Figure 3: Calibration curve of fluorescence of phiLOV

Methodology

Protocol for cloning devices

The devices, as shown in Table 2, were prepared using BioBrick assembly. Parts J23101, J23106, J23117, I13504, I20270 and R0040 were taken from the iGEM distribution plates and each transformed into TOP-10 competent cells. The promoters were digested with Pst1 and Spe1 and the GFP part, I13504, was digested with Xba1 and Pst1. The I13504 part was then ligated into each promoter plasmid and transformed into TOP-10 cells to create the three required devices in pSB1C3 (Figure 4). Restreaks were carried out for one colony of each device and control and three colonies of each (labelled 1, 2 and 3) were picked and grown separately. Sequencing was carried out to check the correct devices had been created.

Table 2: Summary of BioBrick used

Figure 4: Device cloning strategy

Preparation for measurements

Overnight cultures of colony 1-3 of each device were set up (in Luria broth with chloramphenicol) to provide 1ml for measuring on a 96-well plate. As the broth gives off fluorescence samples were also prepared by spinning down cells, in the overnight cultures, to pellets and resuspending in PBS. It was determined the PBS method gave the most accurate measurements so readings were taken using this method for all three biological replicates and technical replicates.

Protocol for measurements

The spectrometer was used to measure absorbance at 600nm of each sample. Samples were then diluted to 0.5 with PBS and rescanned. The Typhoon was used to measure the GFP fluorescence at 475nm of each device and control on a 96 well plate. These methods were repeated for each biological and technical replicate.

The controls

A negative control for background cell fluorescence was included as cells containing the device R0040 but without a promoter, to mimic burden of the promoter. PBS was used to control for media-only background. A positive control for GFP fluorescence was included as cells containing the device I20270, a GFP part with the promoter J23151. In addition in order to obtain absolute values for fluorescence, set standards of FAM oligo were also measured.

Protocol for calculating a conversion factor for absolute units

A dilution series of FAM labelled oligonucleotide was measured (Figure 5) and converted to numerical readings (Table 3) to enable absolute values for the devices to be calculated. The calibration curve (Figure 6) shows a y intercept of 4.79E+06x. Therefore the fluorescence readings of the devices will be divided by the conversion factor of 4,790,000 to give absolute fluorescence as equivalent to pmol of FAM labelled oligonucleotide. Absolute values should be comparable across different equipment and protocols.

Figure 5: Fluorescence readings of a dilution series of FAM labelled oligonucleotide. 10pmol = 10pmol FAM labelled oligonucleotide in 100µl PBS.

Table 3: Fluorescence readings of FAM labelled oligonucleotide.

Figure 6: Confirmation of linear relationship between FAM labelled oligonucleotide concentration and measured fluorescence on the Typhoon. Gradient of this calibration curve is the conversion factor for fluorescence as measured by the Typhoon to equivalent pmol of FAM labelled oligo.

Measurements

Direct Measurement (Raw Data)

The A600 of each device colony 1-3 and technical replicates were measured along with the controls (table 4).

Table 2: Absorbance at 600nm for each biological and technical replicates of the devices and controls. Units are arbitrary.

• Wells – Positive control I20270. Colony 1-3 (horizontal) and replicates 1-3 (vertical)
• Wells – Negative control R0040. Colony 1-3 (horizontal) and replicates 1-3 (vertical)
• Wells – Device 1 J23101. Colony 1-3 (horizontal) and replicates 1-3 (vertical)
• Wells – Device 2 J23106. Colony 1-3 (horizontal) and replicates 1-3 (vertical)
• Wells – Device 3 J23117. Colony 1-3 (horizontal) and replicates 1-3 (vertical)

The fluorescence was also measured (figure 7) and the resulting images converted to numerical readings (table 5).

Figure 7: Fluorescence results of the three devices and the positive and negative controls. A. Shows the image at low brightness to compare the J23101 and J23106 devises. B. Shows the image at high brightness to compare the J23117 device with the two brighter devices.

Table 5: Summary of fluorescence data measured for the three devices and controls.

• Wells – Positive control I20270. Colony 1-3 (horizontal) and replicates 1-3 (vertical)
• Wells – Negative control R0040. Colony 1-3 (horizontal) and replicates 1-3 (vertical)
• Wells – Device 1 J23101. Colony 1-3 (horizontal) and replicates 1-3 (vertical)
• Wells – Device 2 J23106. Colony 1-3 (horizontal) and replicates 1-3 (vertical)
• Wells – Device 3 J23117. Colony 1-3 (horizontal) and replicates 1-3 (vertical)
• Wells – Empty wells.

Derived Measurements (Conversion to Absolute units)

1. The average background absorbance was removed by subtracting the average of the empty wells with no PBS or sample (423,343.279).
2. The average absorbance of control E.coli cells was removed by subtracting the average of the TOP 10 cells with R0040 (222,475).
3. These values were divided by the absorbance values at 600nm to give the fluorescence per OD 600 in arbitrary units (Table 6).
4. Dividing these values by the conversion factor as determined from the FAM oligo dilutions (479,000) gives the absolute fluorescence equivalent to pmol of FAM oligo per A600 of cells (Table 6).

Table 6: Derived measurements of devices and controls.