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

o Sean Colloms - Supervisor for the InterLab study. Helped with cloning devices, taking measurements of the devices and editing the wiki page.
o Vilija Lomeikaite - Set up overnight cultures of the devices
o Ye Yang - Designed and formatted the Interlab wiki page
o Andrey Filipov - Carried out the calibration measurements for the spectrophotometer
o 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.

Detailed Lab Book

o 17/08/2015 (Day 1) - Made TOP-10 competent cells using overnights. Got the parts J23101, J23106, J23117, I13504, I20270 and R0040 off the iGEM distributions plates. Transformed the resuspended DNA into the competent cells, plated them with the appropriate antibiotic and grew them overnight.
o 18/08/2015 (Day 2) - Picked a single colony of each part, inoculated broth and grew over night.
o 19/08/2015 (Day 3) - Carried out mini preps of each part from overnights and made glycerol stocks. Digested promoters J23101, J23106 and J23117 with Pst1 and Spe1 and I13504 with Xba1 and Pst1. J23106 and I13504 didn't cut correctly therefore set up more overnights to repeat digestion.
o 20/08/2015 (Day 4) - Carried out mini preps I13504 and J23106 and re digested each part. Carried out gel extractions, purifications and ligated each promoter to the GFP part I13504. Set up over nights of TOP-10.
o 21/08/2015 (Day 5) - Transformed TOP-10 cells with each device (I13504:J23101, I13504:J23106 and I13504:J23117) and positive/negative controls and plated them.
o 24/08/2015 (Day 6) - Set up overnights and restreaks of 1 colony of each device.
o 25/08/2015 (Day 7) - Prepared samples of each device and controls for measurement by two methods; measuring the A600 of devices in broth and diluting to 0.5 and diluting samples of devices in PBS and measuring their A600 to determine to most accurate method.
o 26/08/2015 (Day 8) - Set up overnights of colony 1,2 and 3 of each device, positive and negative control and technical replicants.
o 27/08/2015 (Day 9) - Carried out mini preps of colony 1 of each device and controls and sent for sequencing. Prepared samples of each device (colony1-3) for measurement along with two technical replicants using the PBS method. Carried out GFP fluorescence readings on a 96 well plate of each device, positive and negative control and a dilution series of LOV proteins and FAM labelled oligonucleoutide.
o 28/08/2015 (Day 10) - Calculated absolute values of fluorescence of GFP and submitted the completed InterLab Worksheet and Protocol forms.
o 31/08/2015 -13/08/2015 – Completed wiki page.

Equipment

Equipment used to acquire measurements

Model and manufacturer:
o Incubator – 2cm shaking diameter
o BioMate™ 3S Spectrophotometer: Life Science Analyzer – 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 a calibration curve (Figure 3) carried out to calibrate the Typhoon. Fluorescent proteins derived from voltage (LOV) domains are smaller and more efficient under anaerobic conditions than green fluorescent proteins (GFP) (Buckley et, al. 2015). iLOV, an improved LOV flavoprotein, was originally engineered as a reporter for viral infection from phototropin, the blue light receptor. We used phiLOV which is a photostable version of the iLOV fluorescence reporter.

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

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.

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 lb broth gave noticeable background fluorescence samples were also prepared by spinning down cells, in the overnight cultures, to pellets and resuspending in PBS (phosphate buffered saline). 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.

The recipe used for a 1 x solution of PBS was 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 dissolved in 800ml of H2O, the pH adjusted to 7.4 and the final volume made up to 1 litre with distilled H2O.

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. A positive control for GFP fluorescence was included as cells containing the device I20270, a GFP part with the promoter J23151. PBS was used to control for media-only background. 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

We used a 6-FAM (6-carboxyfluorescein) labelled oligonucleotide to standardise our fluorescent results. This allowed us to express our GFP levels as equivalent amounts of 6-FAM. 6-carboxyfluorescein is the most commonly used fluorescent dye for labelling oligonucleotides, and therefore should be readily available to most iGEM teams. 6-FAM labelled oligonucleotides can be quantitated by measuring the UV absorbance at 260 nm (measuring the DNA concentration). 6-FAM has similar fluorescent properties to eGFP (excitation peak at 492 nm and an emission maximum of 517 nm for 6-FAM compared to 488 nm excitation and 508 nm emission for E0040 GFP mut3b).

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) has a line gradient of 4.79x10^6. 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.

Sequencing

J23101:I13504

Plasmid Map:

J23106:I13504

Plasmid Map:

J23117:I13504

Plasmid Map:

Measurements

Direct Measurement

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

**Table 4**: Absorbance at 600nm for each biological and technical replicates of the devices and controls. Units are arbitrary.

Derived Measurements

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).

Estimation of absolute number of GFP molecules per cell

We attempted to estimate the absolute number of GFP molecules per cell (Table 7) using our phiLOV results and some simplifying assumptions.

**Table 7**: Summary of absolute number of GFP molecules per cell.

Conclusion

Our measurements have shown that promoters J23101 and J23106 produce more fluorescence with I13504 than the known GFP part I20270 and J23117 shows far less fluoresence, with measurements closer to the negative control R0040 (Figure 8).

**Figure 8** Mean and standard deviation (error bars) for each device and sample.

References

Buckley, A. Petersen, J. Roe, A. Douce, G. Christie, J. (2015). LOV-based reporters for fluorescence imaging. Current Opinion in Chemical Biology. 27 (1), p39–45.