Team:DTU-Denmark/InterLab

InterLab

To quote the official Interlab Measurement Study site, “reliable and repeatable measurement is a key component to all engineering disciplines”.

As a first step in our wetlab efforts we decided to embark on the fifth Interlab Measurement Study. In previous studies, it was shown that the variability between different lab measurements could be reduced by GFP fluorescence expression against a known concentration of a fluorescent molecule [minder det for meget om det der står på siden?]. However, the number of cells in the sample also holds a large impact on the variability.

Therefore, this year’s interlab study is about reducing the variability in fluorescence measurements between labs by normalizing to CFU or absolute cell count. To this, we firstly needed a plate reader that is capable of measuring both absorbance and fluorescence. We used the Biotek Synergy MX plate reader. Specifications of this model important to the protocol in use was:

- It could measure both absorbance and fluorescence
- It had pathlength correction
- Temperature settings went from 4 to 65°C with a precision of ± 0.5°C at 37°C.
- Bandpass width was 530/20
- Excitation was 485 nm
- It used top optics

0. Transformation of competent cells

Luckily, we did not need to create our own competent cells as we already had high-efficiency dh5α cells at hand. As we had a sufficient amount of colonies on our plates, we picked two colonies from each plate and inoculated them in LB medium + chloramphenicol O/N at 37°C and 220 rpm.

1. Calibration 1:​ OD6​00​ Reference point - LUDOX Protocol

Afterwards, we measured the LUDOX CL-X (45% colloidal silica suspension) as a single point reference to gain a conversion factor to transform the absorbance to a OD600 measurement. Plate readers, in general, are volume dependent and this calibration was therefore necessary. We obtained the following data:

LUDOX CL-X H2O
Replicate 1
 0.051 0.036
Replicate 2 0.052 0.035
Replicate 3 0.054 0.037
Replicate 4 0.052 0.037
Arith. Mean 0.052 0.036
Corrected Abs600 0.016
Reference OD600 0.063
OD600/Abs600 3.938

2. Calibration 2:​ Particle Standard Curve - Microsphere Protocol

Next we prepared a dilution series of monodisperse silica microspheres to measure the Abs​600 in our plate reader. Since there is a known amount of particles per volume and their size and optical characteristics are similar to those of cells, we could use the measurement to construct a standard curve. Our results were as following:

Number of Particles 2,35E+08 1,18E+08 5,88E+07 2,94E+07 1,47E+07 7,35E+06 3,68E+06 1,84E+06 9,19E+05 4,60E+05 2,30E+05 0
Replicate 1 0,994 0,575 0,311 0,163 0,101 0,091 0,042 0,04 0,043 0,038 0,036 0,036
Replicate 2 0,97 0,567 0,271 0,162 0,106 0,064 0,051 0,042 0,039 0,038 0,037 0,042
Replicate 3 1,092 0,615 0,28 0,244 0,104 0,082 0,053 0,041 0,039 0,035 0,037 0,038
Replicate 4 1,199 0,454 0,283 0,148 0,096 0,069 0,048 0,041 0,039 0,037 0,036 0,036
Arith. Mean 1,064 0,553 0,286 0,179 0,102 0,077 0,049 0,041 0,04 0,037 0,037 0,038
Arith. Std.Dev. 0,104 0,069 0,017 0,044 0,004 0,012 0,005 0,001 0,002 0,001 0,001 0,003
Arith. Net Mean 1,026 0,515 0,248 0,141 0,064 0,039 0,011 0,003 0,002 -0,001 -0,002



Particles / OD

Number of Particles 2,35E+08 1,18E+08 5,88E+07 2,94E+07 1,47E+07 7,35E+06 3,68E+06 1,84E+06 9,19E+05 4,60E+05 2,30E+05
Mean particles / Abs600 2,29E+08 2,29E+08 2,37E+08 2,08E+08 2,31E+08 1,91E+08 3,50E+08 6,13E+08 4,60E+08 -4,60E+08 -1,53E+08
Mean of med-high levels: 2,19E+08.

3. Calibration 3:​ Fluorescence standard curve - Fluorescein Protocol

In the next segment, we were asked to create a fluorescence standard curve using fluorescein. This was done in order to compare the fluorescence outputs from team to team. As the stability of the GFP protein, as well as the high cost for its purification, is a drawback, we used fluorescein with similar excitation and emission properties as GFP. By creating a dilution series, the following results were gained:

Fluorescein uM 10 5 2,5 1,25 0,625 0,313 0,156 0,078 0,039 0,0195 0,0098 0
Replicate 1 34206 19612 10889 5539 2782 1420 718 341 198 108 34 0
Replicate 2 33672 19429 10913 5399 2736 1398 695 355 175 87 28 0
Replicate 3 32965 18939 10812 5462 2942 1410 713 380 163 95 45 5
Replicate 4 33634 19484 10718 5511 2715 1429 690 368 161 110 65 12
Arith. Mean 33619 19366 10833 5478 2794 1414,3 704 361 174,25 100 43 4,25
Arith. Std.Dev. 508,37 294,81 87,94 61,39 102,72 13,33 13,59 16,79 16,99 10,92 16,27 5,68
Arith. Net Mean 33615 19362 10829 5473,5 2789,5 1410 699,75 356,75 170 95,75 38,75
Fluorescein/a.u.

Fluorescein uM 10 5 2,5 1,25 0,63 0,31 0,16 0,08 0,04 0,02 0,01
uM Fluorescein/a.u. 2,97E-04 2,58E-04 2,31E-04 2,28E-04 2,24E-04 2,22E-04 2,23E-04 2,19E-04 2,30E-04 2,04E-04 2,52E-04
Mean uM fluorescein / a.u.: 2,33E-04. MEFL / a.u.: 1,40E+09.

4. Cell measurement of transformed cells: OD6​00 and fluorescence

After performing the calibrations with a pleasing result, we measured OD6​00 and fluorescence of the transformed cells at 0 and 6 hours. Measurements of given times were created and calculations with the values we obtained from the standard curves and reference points gave us the following data:

By these data, we can conclude that the interlab study was successful and the interlab study protocol can be used for standardization.