InterLab

1. Introduction

PharMARSy decided to participate in the fifth InterLab study. The InterLab study, organized by the measurement committee of iGEM, was an option to obtain the bronze medal requirement #4. The goal of this year’s InterLab study was to collect data to help answer the 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?”

The purpose of the InterLab study is to promote standardization of synthetic biology by creating a solid “measurement tool” for measurements of GFP to be used by the iGEM community but also researchers in general. To read more about the InterLab in general please read more here: https://2018.igem.org/Measurement/InterLab.

1.1 Protocols and data submission

A protocol was provided by the measurement committee which included information of how to carry out the three calibrations and the two experiments that constituted this year´s InterLab study. An Excel sheet was also provided whereto most of the data were to be collected. Information of the details of the experiments and some extra data was handed over to the measurement committee through online submitted forms.

1.2 Plate reader and plates

To measure the absorbance and fluorescence we used the plate reader SpectraMax M5. The 96-well plates used had black sides and a clear, flat bottom. The machine was set to shake for five seconds before the read. As the machine was not able to keep the temperature fixed at a specific temperature, measurements were taken between 20-30 ^oC.

For absorbance measurements, absorbance of the samples were measured at 600 nm. Pathlength correction was set to ‘off’. For fluorescence measurements, samples were measured as a top read with an excitation wavelength at 485 nm and an emission wavelength at 525 nm (cutoff at 515 nm).

2. Calibrations

2.1 Ludox

In this calibration, abs₆₀₀ of LUDOX CL-X (45% colloidal silica suspension) and water was measured. The LUDOX CL-X solution was provided in the InterLab test kit and was not further processed before the measurement.

This calibration was performed to obtain a conversion factor using LUDOX CL-X as a reference point that can be used to convert abs₆₀₀ data into a comparable OD₆₀₀ measurement.

We obtained the following data:

2.2 Silica Beads

In the second calibration the abs₆₀₀ was measured for solutions of silica beads and of water. The silica beads were provided in the InterLab test kit and herefrom a microsphere stock solution was made. This solution was then used to make a dilution series.

This calibration was made to create a standard curve of different microspheres concentrations (which are alike Escherichia coli) to an abs₆₀₀. This makes it possible to convert abs₆₀₀ to a number of cells.

We obtained the following data:

2.3 Fluorescein

In the third calibration, fluorescence (excitation: 485 nm, emission: 525 nm) was measured for fluorescein and of Phosphate Buffered Saline (PBS) with a pH of 7.45.

The fluorescein solution was provided in the InterLab test kit. First, a fluorescein stock solution was made and this solution was then used to make a dilution series.

This calibration was performed to obtain a standard curve of fluorescence specific for our plate reader, and was done to make it possible to compare the data made in these experiments with data from other iGEM teams, as fluorescence values varies broadly with different instruments.

We obtained the following data:

3. Absorbance and fluorescence of transformed cells

In this experiment, bacteria were transformed with plasmids containing different promoters for GFP. After the transformation two colonies were picked and an overnight culture with each colony was made. The overnight culture was diluted to an abs₆₀₀ of 0.02 in a total volume of 12 mL. The absorbance and fluorescence were then measured of this solution before (t=0 hours) and after incubation (t=6 hours).

Luria Bertani (LB) media was used throughout the experiment and the antibiotic used as selective marker was chloramphenicol (CAM).

3.1 Plasmids of transformants

The bacteria used in this experiment was Escherichia coli strain DH5α. Using the iGEM Parts Distribution Kit Plates, eight transformations were made containing the following:

3.2 Absorbance

Absorbance at 600 nm was measured for two colonies for each combination of test device or control, and for each colony, four technical replicates were measured. We obtained the following data:

3.3 Fluorescence

We obtained the following data:

4. Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures

In this experiment, E. coli was transformed with the following plasmids:

• Negative control BBa_R0040 Kit Plate 7 Well 2D
• Positive control BBa_I20270 Kit Plate 7 Well 2B

Two colonies were selected after each transformation and used for overnight cultures.

Step one: Starting sampling preparation

The overnight cultures were diluted and the OD₆₀₀ was measured. Using this data, the solution was further diluted to an OD₆₀₀=0.1. This was done in triplicate for each culture. It was measured for all samples whether an OD₆₀₀=0.1 was obtained. The following OD₆₀₀ was measured (supposed to be 0.1):

Step two: Dilution series

A dilution series was then made using with the starting samples. 100 µl of dilution number 3, 4 and 5 were plated and grown overnight.

Step three: CFU/mL/OD Calculation Instructions

The colonies on the plates were counted and CFU per 1mL of an OD₆₀₀ = 0.1 culture were calculated. We obtained the following data:

5. Conclusion

The data was submitted to the measurement committee the 24^th of July and was accepted by the measurement committee the 30^th of July.

Completing the InterLab studies served as a good introduction for us to be self reliant in the laboratory and train basic laboratory skills. Moreover, we learned our way around the laboratory and our facilities before starting the project.

We think that the idea behind the InterLab study is great and we look forward to see the other groups’ results and the final conclusion of the collected data from the iGEM HQ.