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Revision as of 16:33, 15 October 2018

InterLab

Reliable and repeatable measurement is a key component to all engineering disciplines.- iGEM
Placeholder


Introduction

As a kickoff for our work in the lab, our team decided to take part in this year’s international InterLab Measurement Study, intending to improve measurement tools and to develop a solid and repeatable measurement protocol, making the comparison of measurements carried out in different labs more straightforward. Since being one of the most utilized markers all over the world, the fluorescent protein GFP (Green Fluorescent Protein) was chosen as a measurement marker.

In order to contribute to the 2018 InterLab Measurement study, the participating teams were advised to measure the OD600 and the fluorescence of 8 different constructs expressed in DH5a E.colis on a plate reader. Additionally, a protocol to calibrate OD600 to colony forming units (CFU) counts, which directly refer to the cell concentration in the culture, was provided.

Materials and Methods

Materials

Plate Reader: Tecan Infinite M200 Pro

96 well plates (white): Source unknown

DH5a Escherichia Coli

Glass Test Tubes

Devices

Table 1 Devices that were transformed during the InterLab Study and their corresponding iGEM Part Numbers.
DevicePart Number
Positive ControlBBa_I20270
Negative ControlBBa_R0040
Test Device 1BBa_J364000
Test Device 2BBa_J364001
Test Device 3BBa_J364002
Test Device 4BBa_J364007
Test Device 5BBa_J364008
Test Device 6BBa_J364009

Workflow Cell Measurements

Figure 1 Workflow of the cell measurements.
Figure 1 Workflow of the cell measurements.

Measurements

Calibration Measurements

All calibration measurements were performed as suggested by the protocol.

OD600 Reference Point– Ludox CL-X

Table 2 Results of the LUDOX Cl-X Calibration Measurement
Ludox CL-XH20
Replicate 10.05610.0389
Replicate 20.05680.0393
Replicate 30.05950.0385
Replicate 40.05660.0388
Arith. Mean0.0570.039
Corrected Abs6000.018
Reference OD6000.063
OD600/Abs6003.429

Particle Standard Curve – Microspheres

Figure 2.1 Microsphere particle standard curve on a linear scale
Figure 2.1 Microsphere particle standard curve on a linear scale
Figure 2.2 Microsphere particle standard curve on a logarithmic scale
Figure 2.2 Microsphere particle standard curve on a logarithmic scale

Fluorescence Standard Curve – Fluorescein

Figure 3.1 Fluorescent standard curve on a linear scale
Figure 3.1 Fluorescent standard curve on a linear scale
Figure 3.2 Fluorescent standard curve on a logarithmic scale
Figure 3.2 Fluorescent standard curve on a logarithmic scale


Cell Measurements

Transformation of 8 Test Devices

The transformation was carried out according to the iGEM Protocol. The clones were picked and treated as suggested by the cell measurement workflow (Figure 1).

OD600 Measurement

The OD600 of all samples was measured at 0h and after 6h of incubation at 37°C. All samples started with similar ODs of about 0.03 to 0.04. After 6h an increase of the OD was noticeable in all strains. The negative control (LB + Amp) did not show a change of OD within the course of the experiment (Figure 4).
Figure 4 Measurement of the OD600 of the 8 transformed strains after 0h and after 6h of incubation
Figure 4 Measurement of the OD600 of the 8 transformed strains after 0h and after 6h of incubation

Fluorescence Measurement

Furthermore, the fluorescence of the strains with the 8 different devices was measured after 0h and after 6h of incubation. The negative control showed, as expected, very low levels of fluorescence, whereas the positive control showed in comparison to that, much more fluorescence. However, contrary to our expectations, the positive control showed less fluorescence after 6h as at time point 0. The same is true for the devices 3, 4 and 5. However, the values of the devices 3 and 5 correspond to the negative control, from which it can be concluded that these devices do not show fluorescent activity. In our experiment, the devices 1 and 2 show the highest fluorescent values after 6h of incubation.
Figure 5 Fluorescence measurement of the 8 different strains after 0h and 6h of incubation
Figure 5 Fluorescence measurement of the 8 different strains after 0h and 6h of incubation

CFU Protocol

Hier ist ein Text. Lol Lol Lol.

Discussion

During the performance of the fluorescent measurement of the cells, it became clear, that the utilized plate reader did not work properly. For this reason, we had to repeat the fluorescent measurements with a different plate reader on a different day. However, we tried to make sure, that all measurements still are as comparable as possible by using the same plate reader model and the same plates. After sending in our data and informing iGEM of the difficulties we were encountering, we received the message, that even though our data does not match the iGEM measurement standards, we will still be awarded with the Bronze medal standard for our many tries to handle the problems correctly. For this, we want to thank the iGEM committee very much.