Team:Tuebingen/InterLab

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InterLab

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

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.

Device	Part Number
Positive Control	BBa_I20270
Negative Control	BBa_R0040
Test Device 1	BBa_J364000
Test Device 2	BBa_J364001
Test Device 3	BBa_J364002
Test Device 4	BBa_J364007
Test Device 5	BBa_J364008
Test Device 6	BBa_J364009

Workflow 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-X	H20
Replicate 1	0.0561	0.0389
Replicate 2	0.0568	0.0393
Replicate 3	0.0595	0.0385
Replicate 4	0.0566	0.0388
Arith. Mean	0.057	0.039
Corrected Abs600	0.018
Reference OD600	0.063
OD600/Abs600	3.429

Particle Standard Curve – Microspheres

Figure 2.1 Microsphere particle standard curve on a linear 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 3.2 Fluorescent standard curve on a logarithmic scale

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

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

Colony Forming Units Protocol

The CFU protocol is a way to detect the proportion of living cells in an OD₆₀₀ measurement. It is assumed that a living cell produces one colony. This allows a direct conclusion to be drawn as how many living cells are contained in a certain volume. For this purpose, the ODs of the postcultures of the positive and negative controls of the Cell Measurement Protocol were measured and diluted in triplicates to an OD₆₀₀ of 0.1 with LB medium and chloramphenicol. The obtained triplicates were verified with a photometer. The 12 preparations (2 positive + 2 negative in 3 preparations each) were then diluted using the following pipetting schemes:

Figure 6 Dilution Scheme for the CFU protocol

Of these 5 dilutions of the 12 preparations, the dilutions 3, 4, and 5 were spread with 100µl each on LB-agar + chloramphenicol. After overnight incubation at 37°C the following colony numbers were obtained:

Colony count on LB+CM-Plates

Table 1: Colonies of positive control 1

	Replicate 1	Replicate 2	Replicate 3
Dilution 3	676	2036	2488
Dilution 4	212	504	252
Dilution 5	37	97	131

Table 2: Colonies of positive control 2

	Replicate 1	Replicate 2	Replicate 3
Dilution 3	2376	1216	1744
Dilution 4	204	648	300
Dilution 5	15	52	34

Table 3: Colonies of negative control 1

	Replicate 1	Replicate 2	Replicate 3
Dilution 3	1320	1296	1672
Dilution 4	400	388	296
Dilution 5	48	80	61

Table 4: Colonies of negative control 2

	Replicate 1	Replicate 2	Replicate 3
Dilution 3	2440	3448	3216
Dilution 4	1172	1520	1488
Dilution 5	252	248	334

Based on these colony numbers, the living cell count of the overnight cultures could now be finally calculated.

Table 5: Calculated amount of living cells

	Positive 1	Positive2	Negative 1	Negative 2
Replicate 1	40,36	26,56	76,86	584,66
Replicate 2	184,36	67,66	153,66	574,46
Replicate 3	248,96	44,26	117,16	774,96

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.

I

• Introduction
• Materials
  and Methods
• Measurements
• Discussion