Team:Chalmers-Gothenburg/InterLab

Chalmers Gothenburg iGEM 2017

Introduction

The Interlab study aims to improve the tools for measurements that are available in both the iGEM community but also the whole synthetic biology community. One of the biggest challenges in synthetic biology is the fluorescence data that is often hard to compare since it is reported in different units or that the data is processed in different ways. The goal of the InterLab study is to address this issue by providing researchers with a detailed protocol and data analysis form for measuring GFP in a plate reader. This is done by comparing results obtained by various iGEM teams in order to quantify the expression of different constructs with fluorescence, both with a positive and a negative control.

In the iGEM interlab study of 2017, 6 different fluorescent protein expression plasmids were tested; J364000, J364001, J364002, J364003, J364004, J364005, a positive I20270, and a negative R0040 control plasmid. The plasmids were transformed into DH5α E.coli with the team’s own transformation protocol. All of the fluorescence devices that were tested were in the pSB1C3 plasmid backbone and the transformed E.coli were streaked and cultured on/in LB+chloramphenicol. To measure the difference in expression of fluorescence by the different plasmids, a plate reader that both measured the absorbance and fluorescence intensity was used.

Results

The results from the measurements are shown below. First the results for the OD reference point and the fluorescein standard curve is shown. After that the results from the main experiments is presented.

OD reference point

The OD at wavelength 600nm was measured for LUDOX-HS40 and water to get OD reference point for the following OD measurements, see Table 1.

Table 1. OD measurements of LUDOX-HS40 and water.
LUDOX-HS40 H2O
Replicate 1 0,031 0,022
Replicate 2 0,030 0,023
Replicate 3 0,034 0,024
Replicate 4 0,032 0,024
Arith. Mean 0,032 0,023
Corrected Abs600 0,009
Reference OD600 0,043
OD600/Abs600 5

Fluorescein Standard Curve

Different concentrations of fluorescein was measured with four replications in order to be able to relate the GFP expression to a concentration for the following fluorescence measurements in the main experiment. Below the standard curve of the measured values can be seen, both with the actual values and logarithmed.

Fluorescein standrad curve
Figure 1. The Fluorescein standard curve was measured and prepared using 1x Fluorescein and PBS in a serial dilution on day 2.

Fluorescein Standard Curve (log scale)

Fluorescein standrad curve (log scaled)
Figure 2. The Fluorescein standard curve in log scale.

OD measurements

OD measurements
Figure 3. Absorbance of the six different devices together with one negative, one positive control and the blank. The values of the devices are an averages of the the two colonies for each device and their four replicates.

Fluorescence measurements

Fluorescence measurements
Figure 4. Fluorescence of the six different devices together with one negative, one positive control and the blank. The values of the devices are an averages of the the two colonies for each device and their four replicates.