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<h2 style="color:#F8A05B;">Calibration 1:​ OD6​00​ Reference point - LUDOX Protocol</h2>
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<li><a href="#il216">21/6/2018</a></li>
 
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<li><a href="#il226">22/6/2018</a></li>
 
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<h4 id="il216">Thursday 21/6/2018</h4>
 
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Competent cells were prepared for the interlab studies using the given the <a target="_blank" href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf">iGem 2018 interlab platereader protocol</a>. We did not need to create our own competent cells as we already had high-efficiency dh5α cells ready.</p>
 
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<h4 id="il226">Friday 22/6/2018</h4>
 
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Today we transformed our prepared competent cells by using the <a target="_blank" href="http://parts.igem.org/Help:Protocols/Transformation">iGem 2018 interlab study transformation</a>.</p>
 
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<h4 id="il266">Tuesday 26/6/2018</h4>
 
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Luquid LB media was prepared using the "Luria-Bertani (LB) Medium Preparation" protocol for the interlab studies in the
 
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morning, as we did not have enough for the next step. Interlab is put on hold until we have a platereader.</p>
 
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<h4 id="il27">Monday 2/7/2018</h4>
 
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We picked 2 colonies from each of our transformation plates and inoculated them in the apropriate LB and
 
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Chloramphenicol medium. The cells were left to grow overnight at 37 degrees Celsius.</p>
 
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<h4 id="il37">Tuesday 3/7/2018</h4>
 
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Today we finally had the platereader available and continued on the protocol from the 21st of June. We encountered som problems with our fluorescein samples and decided to redo them. We also decided to redo the overnight
 
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cultures as we would not have the time to use them today</p>
 
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<h4 id="il47">Wednesday 4/7/2018</h4>
 
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As we finally had all our controls finished, we cpntinued with the "Cell measurement" protocol. Our initial absorbance for
 
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our samples were way too high. We then finalized this part of the study and continued with the "Colony Forming Units" protocol.</p>
 
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<h4 id="il57">Thursday5/7/2018</h4>
 
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Data was extracted from our platereader and this, together with the CFU data, was sent off to finalize the interlab study.
 
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Another thing on our long list that can be ticked off.</p>
 
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Revision as of 19:15, 12 October 2018

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. 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

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.

Fig. 1a: - LB-CAM control (100 py). Fig. 1b: - Starting the overnight inoculation.

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

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:

Mean of med-high levels: 2,19E+08.

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:

Mean uM fluorescein / a.u.: 2,33E-04. MEFL / a.u.: 1,40E+09.

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:

Flourescence Raw Readings

Hour 0: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 47 40 141 74 13 140 102 35 15
Colony 1, Replicate 2 0 35 161 76 25 140 78 37 38
Colony 1, Replicate 3 3 69 84 85 26 115 71 46 19
Colony 1, Replicate 4 3 26 87 65 25 114 68 54 45
Colony 2, Replicate 1 24 32 139 131 40 78 114 46 15
Colony 2, Replicate 2 43 53 145 103 24 92 92 80 26
Colony 2, Replicate 3 16 30 117 62 23 83 85 62 5
Colony 2, Replicate 4 2 37 105 115 16 92 72 42 27
Hour 6: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 9 412 196 744 32 360 120 265 39
Colony 1, Replicate 2 28 385 235 769 29 364 197 309 24
Colony 1, Replicate 3 30 399 264 840 10 409 170 339 29
Colony 1, Replicate 4 0 423 276 803 21 419 150 317 17
Colony 2, Replicate 1 15 500 304 754 43 971 159 342 20
Colony 2, Replicate 2 37 469 319 778 20 905 162 316 9
Colony 2, Replicate 3 36 522 325 846 39 931 144 333 11
Colony 2, Replicate 4 38 535 286 864 14 968 167 388 29

Abs600 Raw Readings

Hour 0: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 0,062 0,07 0,058 0,054 0,059 0,054 0,052 0,054 0,04
Colony 1, Replicate 2 0,058 0,061 0,063 0,058 0,063 0,058 0,05 0,054 0,041
Colony 1, Replicate 3 0,065 0,065 0,052 0,053 0,055 0,054 0,046 0,058 0,04
Colony 1, Replicate 4 0,056 0,057 0,053 0,055 0,056 0,079 0,059 0,053 0,04
Colony 2, Replicate 1 0,063 0,061 0,067 0,061 0,064 0,054 0,053 0,057 0,039
Colony 2, Replicate 2 0,063 0,066 0,058 0,061 0,062 0,056 0,049 0,052 0,039
Colony 2, Replicate 3 0,072 0,058 0,059 0,056 0,066 0,054 0,052 0,063 0,039
Colony 2, Replicate 4 0,066 0,06 0,052 0,059 0,062 0,051 0,048 0,06 0,041
Hour 6: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 0,373 0,318 0,062 0,331 0,361 0,083 0,054 0,339 0,042
Colony 1, Replicate 2 0,393 0,345 0,069 0,353 0,357 0,083 0,058 0,35 0,04
Colony 1, Replicate 3 0,388 0,344 0,065 0,361 0,356 0,089 0,059 0,346 0,039
Colony 1, Replicate 4 0,377 0,334 0,066 0,338 0,359 0,091 0,058 0,368 0,043
Colony 2, Replicate 1 0,404 0,397 0,08 0,326 0,362 0,234 0,057 0,365 0,039
Colony 2, Replicate 2 0,37 0,331 0,082 0,295 0,339 0,219 0,056 0,324 0,039
Colony 2, Replicate 3 0,386 0,335 0,077 0,299 0,37 0,218 0,057 0,346 0,04
Colony 2, Replicate 4 0,402 0,379 0,079 0,33 0,364 0,23 0,056 0,376 0,04
Unit Scaling Factors:
OD600 / Abs600 3,94
uM Fluorescein / a.u. 2,33E-04

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