Team:UNOTT/InterLab





INTERLAB STUDY:

Introduction

Our team decided to take part in the iGEM interlab study this year. This study is organised by iGEM and is aimed at tackling a prominent issue regarding experimental fluorescent reporter data. At present, experiments using fluorescent reporters are almost incomparable; separate groups often interpret fluorescence measurements in largely different ways, making it hard to properly compare results. To combat this, the interlab study is performed each year, iteratively refining an optimal protocol for fluorescence measurement. Theoretically, this would be followed by anyone and yield comparably robust units of fluorescence, facilitating more meaningful comparisons of data between researchers.

The study required us to measure a number of different fluorescent test devices sent to us by iGEM HQ, we followed their protocol and then shared the results we obtain. Teams around the world do the same; collectively, we provide a large set of data illustrating how similar the results were, potentially highlighting any aspects in which the protocol could be improved and, hopefully, showing that this is possible. This is quite an important issue, we felt we should participate and help in any way we could!

Methods

We were given 8 specimens to measure, termed “test devices”. These included a positive (BBa_I20270) and negative(BBa_R0040) control, and 6 different test devices (BBa_J36400, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005).

First we transformed these into DH5α-E. coli cells, then selected two resulting colonies to be prepared for measurement. The two colonies were grown overnight.

But before the experiment could be undertaken, we needed to calibrate our equipment by performing standard reference measurements, enabling the conversion of our data to absolute values. Firstly, we measured a solution of half LUDOX-S40, half water; fluorescein was then serially diluted by half 10 times and measured to give a standard curve.

After 18 hours of growth, the test device sample's optical density was measured, a new batch of LB/Chloramphenicol was inoculated to produce an OD of 0.02. These were then grown at 37°C, OD600nm and fluorescence were measured at 0, 2, 4, and 6 hours.

For a full description of the methods, please visit the official interlab study page

Results

LUDOX-S40 and Fluoroscein Reference Measurements

LUDOX-HS40 H20
Replicate 1 S0.041 0.029
Replicate 2 0.045 0.03
Replicate 3 0.043 0.032
Replicate 4 0.041 0.032
Arith. Mean 0.0425 0.03075
Corrected Abs600 0.01175
Reference OD600 0.0425
OD600/Abs600 3.617
uM Fluoroscein/a.u 50.002512.56.253.131.560.780.390.20.10.050
Replicate 1 261721741310315606298815347954072231247729
Replicate 2 2602117369102995641299715297884102241247830
Replicate 3 2614117333103215602297915277874092251267830
Replicate 4 2624317204102735570296615237894172161267628
Arith. Mean 26144173301030656052983152878904112221257729
Arith. Std. Dev. 92.5989.9925.7429.04413.224.573.594.344.081.150.960.96
uM Fluoroscein/a.u 50.002512.56.253.131.560.780.390.20.10.05
Mean uM Fluorescein/a.u.(×10^-3) 1.91.41.21.1110.990.950.880.780.63
Mean of med-high levels 0.001168

Serial dilution of fluorescein intesnity plotted on linear scaleFluorescein dilution of fluorescein intensity plotted on log (scale Graph)

OD600nm and Fluorescence Results

Fluorescence Data
(colonies averaged and minus background)
Timepoints (Hours)
Test Device0H2H4H6H
Negative Control24.252119.3818.75
Positive27.838.681.9107.5
144.2562.4103.5174.8
22840.193.5137.4
32421.420.521.6
42836.579.6124
52528.440.545.1
623.1219.7519.4
OD600nm Data (colonies averaged and minus background)
Test Device0H2H4H6H
Negative Control0.010.070.30.55
Positive control0.010.060.260.55
10.010.020.030.07
20.0010.050.220.5
30.070.060.250.48
40.010.030.130.4
50.080.050.230.54
60.080.610.250.5

OD600nm measurements of all test devices (minus background), biological replicates were averaged.Fluoresence measurements of all test devices (minus background), biological replicates were averaged.

Graph depicting each test device's averaged fluorescence intensity minus background
Graph depicting each test device's averaged OD600nm minus background

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