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Team:Edinburgh/Part Collection

InterLab Study

Introduction

The term InterLab stands for Inter-Laboratory studies. The iGEM InterLab studies conducted during the last two years have been of huge importance to establish a standard for reproducibility of fluorescent measurements. The measurement of fluorescence is an essential tool in synthetic biology. However, the absence of absolute units and standard protocols makes it harder to interpret data and share biological parts between laboratories. The 2016 iGEM InterLab consists of two protocols for measuring GFP that will lead to the creation of absolute units. This collection of measurements can then be used to establish the accuracy with which GFP is measured around the world and what needs to be improved when following a standard protocol. As a team, we decided that the iGEM InterLab was a great opportunity to improve our laboratory skills and take part into an international study.

Plate Reader

All other protocols used in the process of the study can be found on the protocols page.

The aim of the 2016 iGEM InterLab was to quantify the expression of GFP under different promoters and Ribosomal Binding Regions (RBRs). The use of GFP to indirectly measure the expression of a specific protein in cells is a key approach in biology. It enables us to monitor expression levels without killing the cells. The ability measure expression levels per cell is important to be able to quantify strength of promotors. In order to achieve this, population size is indirectly measured using OD600. Expression per cell is then obtained by Fluorescence/OD600.

Both Fluorescence and OD600 are relative measurements, meaning the instruments used and their settings play a crucial role.

To calibrate the instrument and transform relative measurements into absolutes ones, FITC is used as reference material to construct a standard calibration curve for fluorescence. FITC standard is constructed by serial dilution. The resulting FITC curve is used in combination with another FITC standard curve and a GFP standard curve provided by iGEM.

OD600 is a measure of light scattering, therefore light detection is dependent on the physical geometry of the machine. Calibration experiments using a flow cytometer do not involve standard curves. In order to give uniformity to Fluorescence/OD600 data, a standard scattering solution of a mono-dispersed silica suspension (LUDOX) is used.

Method Overview

First the 5 plasmids (Positive Control, Negative Control, Test Device 1, Test Device 2, and Test Device 3) from the InterLab Measurement Kit were transformed into TOP10 competent cells. These were then inoculated into liquid culture and used to do make a MiniPrep. The MiniPrep products were then used for the experiment according to the Plate Reader Protocol.

Protocol

Competent Cells: http://parts.igem.org/Help:Protocols/Competent_Cells

Transformation: http://parts.igem.org/Help:Protocols/Transformation

MiniPrep: http://parts.igem.org/Help:Protocols/Miniprep

Plate Reader Protocol: https://static.igem.org/mediawiki/2016/c/c5/InterLab_iGEM2016_Plate_Reader_Protocol_Updated_July.pdf

Improvements to the Protocol

LUDOX 100% and H2O measurements of 1 ml aliquots were taken using a… Spectrophotometer, figure 1. The reference OD600 and the correction factor was 0.01475 and 2.977693 respectively.

Figure1: OD600 reference point

A 10x dilution of FITC stock solution was diluted in half using 1xPBS following the instructional diagram in the Plate Reader Protocol. A FITC calibration curve was obtained, figure 2.

Figure 2: FITC calibration curve

OD600 of the overnight cultures was measured. Cultures were then diluted with LB medium + chloramphenicol to reach a target OD600 of 0.02. Aliquots of 10 ml of this mixtures were then distributed among the different cultures. 1 ml samples of each device were taken at 0, 1, 2, 3, 4, 5 and 6 hours of incubation at 37ºC and 220 rpm, figure 3.

Figure 3: Abs600 over time

After the 6 hour incubation period, 100 µl of each sample was pipetted in a 96 well-plate following the instructions of Figure 2 from the Plater Reader Protocol. The samples were measured using a…plate reader utilizing the same setting used to create our FITC standard curve, figure 4. The gain was 1000.

Figure 4: Fluorescence over time

A ratio of Fluorescence/Abs600 was then calculated for all devices and controls. The Mean and Standard Deviation were calculated for each of the replicates, figure 5.