Difference between revisions of "Team:DTU-Denmark/InterLab"
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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: | 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: | ||
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Next we prepared a dilution series of monodisperse silica microspheres to measure the Abs600 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: | Next we prepared a dilution series of monodisperse silica microspheres to measure the Abs600 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: | ||
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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: | 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: | ||
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Fluorescein/a.u. | Fluorescein/a.u. | ||
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After performing the calibrations with a pleasing result, we measured OD600 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: | After performing the calibrations with a pleasing result, we measured OD600 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: | ||
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By these data, we can conclude that the interlab study was successful and the interlab study protocol can be used for standardization. | By these data, we can conclude that the interlab study was successful and the interlab study protocol can be used for standardization. | ||
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<h4 id="il216">Thursday 21/6/2018</h4> | <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> | 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> | <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> | 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> | <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 | Luquid LB media was prepared using the "Luria-Bertani (LB) Medium Preparation" protocol for the interlab studies in the | ||
morning, as we did not have enough for the next step. Interlab is put on hold until we have a platereader.</p> | 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> | <h4 id="il27">Monday 2/7/2018</h4> | ||
<hr> | <hr> | ||
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We picked 2 colonies from each of our transformation plates and inoculated them in the apropriate LB and | We picked 2 colonies from each of our transformation plates and inoculated them in the apropriate LB and | ||
Chloramphenicol medium. The cells were left to grow overnight at 37 degrees Celsius.</p> | 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> | <h4 id="il37">Tuesday 3/7/2018</h4> | ||
<hr> | <hr> | ||
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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 | 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 | ||
cultures as we would not have the time to use them today</p> | 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> | <h4 id="il47">Wednesday 4/7/2018</h4> | ||
<hr> | <hr> | ||
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As we finally had all our controls finished, we cpntinued with the "Cell measurement" protocol. Our initial absorbance for | As we finally had all our controls finished, we cpntinued with the "Cell measurement" protocol. Our initial absorbance for | ||
our samples were way too high. We then finalized this part of the study and continued with the "Colony Forming Units" protocol.</p> | 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> | <h4 id="il57">Thursday5/7/2018</h4> | ||
<hr> | <hr> | ||
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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. | Data was extracted from our platereader and this, together with the CFU data, was sent off to finalize the interlab study. | ||
Another thing on our long list that can be ticked off.</p> | Another thing on our long list that can be ticked off.</p> | ||
Revision as of 16:07, 4 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 [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: OD600 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 Abs600 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 |
4. Cell measurement of transformed cells: OD600 and fluorescence
After performing the calibrations with a pleasing result, we measured OD600 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.