This year, we contributed to the competition by participating in the 5th iGEM International Laboratory Study. As with previous years, the goal of the study is to standardize measurement techniques so that synthetic biology labs all over the world can reference and compare collected data reliably. This year’s focus is on converting fluorescence readings into absolute cell counts instead of relying on OD measurements, which can often vary from lab to lab. The materials required to perform this year’s interlab study include a plate reader capable of measuring fluorescence and competent E. coli DH5-alpha. We used a BMG labtech Clariostar plate reader along with black 96-well plates with transparent bottoms, and prepared DH5-alpha in glycerol stock. Using the rest of the required materials found in the iGEM interlab kit, we were able to test 8 plasmids consisting of a positive control, a negative control and 6 test constructs. We first performed calibrations to obtain standard values for our data.
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| − | Our first calibration uses LUDOX CL-X solution to convert absorbance (ABS<sub>600</sub>) to an equivalent measurement of optical density ( | + | Our first calibration uses LUDOX CL-X solution to convert absorbance (ABS<sub>600</sub>) to an equivalent measurement of optical density (OD 600). This is necessary because absorbance measurements are dependent on the depth of the fluid sample, while OD 600 values obtained from a standard spectrophotometer depend only on cuvette width, which is constant. This calibration eliminates measurement variations specific to our plate reader and returns standard values of optical density that other labs can reference. |
</p> | </p> | ||
</div> | </div> | ||
| Line 642: | Line 642: | ||
Figure 2: The curve describing the relationship between particles and Abs<sub>600</sub> in logarithm scale. | Figure 2: The curve describing the relationship between particles and Abs<sub>600</sub> in logarithm scale. | ||
</div> | </div> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/2/26/T--NCTU_Formosa--Close.png" class="close_1"> | ||
</div> | </div> | ||
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Figure 4: The curve describing the relationship between fluorescence and fluorescein concentration in logarithm scale. | Figure 4: The curve describing the relationship between fluorescence and fluorescein concentration in logarithm scale. | ||
</div> | </div> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/2/26/T--NCTU_Formosa--Close.png" class="close_2"> | ||
</div> | </div> | ||
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<div class="sec sec_5"> | <div class="sec sec_5"> | ||
<p class="title_2">Cell Measurement</p> | <p class="title_2">Cell Measurement</p> | ||
| − | <div class="text"><p> Once our calibrations were complete, we moved on to the cell measurement protocol. First we transformed competent DH5-alpha with the 8 plasmids provided in the kit. After incubating for 16 hours at 37 degrees Celsius and 220 rpm, we diluted the solutions to an Abs<sub>600</sub> value of 0.02 before measuring both | + | <div class="text"><p> Once our calibrations were complete, we moved on to the cell measurement protocol. First we transformed competent DH5-alpha with the 8 plasmids provided in the kit. After incubating for 16 hours at 37 degrees Celsius and 220 rpm, we diluted the solutions to an Abs<sub>600</sub> value of 0.02 before measuring both OD 600 as well as fluorescence at 0 hours and 6 hours. Then, using our previous calibrations we converted fluorescence per OD to fluorescence per particle.</p></div> |
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</table> | </table> | ||
</div> | </div> | ||
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</div> | </div> | ||
<img src="https://static.igem.org/mediawiki/2018/2/24/T--NCTU_Formosa--Seemore2.png" class="seemore_3"> | <img src="https://static.igem.org/mediawiki/2018/2/24/T--NCTU_Formosa--Seemore2.png" class="seemore_3"> | ||
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<div class="sec sec_6"> | <div class="sec sec_6"> | ||
<p class="title_2">Colony Formig Units</p> | <p class="title_2">Colony Formig Units</p> | ||
| − | <div class="text"><p> Finally, we test whether our cell measurement conversion was accurate. Because our conversions are based on the calibrations we performed using the silica beads, we observe actual colony forming units on plates and compare with our theoretical conversions. To do this, we incubated 2 samples of both positive and negative control overnight, then diluted 1 mL of the culture to an | + | <div class="text"><p> Finally, we test whether our cell measurement conversion was accurate. Because our conversions are based on the calibrations we performed using the silica beads, we observe actual colony forming units on plates and compare with our theoretical conversions. To do this, we incubated 2 samples of both positive and negative control overnight, then diluted 1 mL of the culture to an OD 600 value of 0.1 and prepared a series dilution. We then spread the final 3 dilutions of each sample onto plates and counted the CFUs. Because we started with an OD 600 value of 0.1, we know the OD values of the subsequent dilutions and can compare the resulting number of CFUs to our conversions from our cell measurement protocol.</p></div> |
<div class="show_4"> | <div class="show_4"> | ||
<div class="table"> | <div class="table"> | ||
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<p class="explanation"> | <p class="explanation"> | ||
<svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-down" class="svg-inline--fa fa-arrow-circle-down fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M504 256c0 137-111 248-248 248S8 393 8 256 119 8 256 8s248 111 248 248zm-143.6-28.9L288 302.6V120c0-13.3-10.7-24-24-24h-16c-13.3 0-24 10.7-24 24v182.6l-72.4-75.5c-9.3-9.7-24.8-9.9-34.3-.4l-10.9 11c-9.4 9.4-9.4 24.6 0 33.9L239 404.3c9.4 9.4 24.6 9.4 33.9 0l132.7-132.7c9.4-9.4 9.4-24.6 0-33.9l-10.9-11c-9.5-9.5-25-9.3-34.3.4z"></path></svg> | <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-down" class="svg-inline--fa fa-arrow-circle-down fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M504 256c0 137-111 248-248 248S8 393 8 256 119 8 256 8s248 111 248 248zm-143.6-28.9L288 302.6V120c0-13.3-10.7-24-24-24h-16c-13.3 0-24 10.7-24 24v182.6l-72.4-75.5c-9.3-9.7-24.8-9.9-34.3-.4l-10.9 11c-9.4 9.4-9.4 24.6 0 33.9L239 404.3c9.4 9.4 24.6 9.4 33.9 0l132.7-132.7c9.4-9.4 9.4-24.6 0-33.9l-10.9-11c-9.5-9.5-25-9.3-34.3.4z"></path></svg> | ||
| − | Table 20: The CFU | + | Table 20: The CFU results of different samples. |
</p> | </p> | ||
</caption> | </caption> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <th><p>Sample</p></th> | ||
| + | <th><p>CFU Concerntration</p></th> | ||
| + | </tr> | ||
| + | </thead> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td> | + | <td><p style="white-space: nowrap;">Positive Colony 1 Triplicate 1</p></td> |
| − | + | <td><p>1.8E+07 CFU/mL</p></td> | |
| − | + | ||
| − | <td> | + | |
| − | + | ||
| − | + | ||
</tr> | </tr> | ||
| − | + | <tr> | |
| − | + | <td><p style="white-space: nowrap;">Positive Colony 1 Triplicate 2</p></td> | |
| − | + | <td><p>2.56E+07 CFU/mL</p></td> | |
| − | <td> | + | |
| − | + | ||
| − | + | ||
</tr> | </tr> | ||
| − | + | <tr> | |
| − | + | <td><p style="white-space: nowrap;">Positive Colony 1 Triplicate 3</p></td> | |
| − | + | <td><p>5.6E+06 CFU/mL</p></td> | |
| − | <td> | + | |
| − | + | ||
| − | + | ||
</tr> | </tr> | ||
| − | + | <tr> | |
| − | + | <td><p style="white-space: nowrap;">Positive Colony 2 Triplicate 1</p></td> | |
| − | + | <td><p>7.44E+07 CFU/mL</p></td> | |
| − | <td> | + | |
| − | + | ||
| − | + | ||
</tr> | </tr> | ||
| − | + | <tr> | |
| − | + | <td><p style="white-space: nowrap;">Positive Colony 2 Triplicate 2</p></td> | |
| − | + | <td><p>1.04E+07 CFU/mL</p></td> | |
| − | + | ||
| − | <td> | + | |
| − | + | ||
| − | + | ||
</tr> | </tr> | ||
| − | + | <tr> | |
| − | + | <td><p style="white-space: nowrap;">Positive Colony 2 Triplicate 3</p></td> | |
| − | + | <td><p>2.4E+06 CFU/mL</p></td> | |
| − | + | ||
| − | <td> | + | |
| − | + | ||
| − | + | ||
</tr> | </tr> | ||
| − | + | <tr> | |
| − | + | <td><p style="white-space: nowrap;">Negative Colony 1 Triplicate 1</p></td> | |
| − | + | <td><p>2.23E+07 CFU/mL</p></td> | |
| − | + | ||
| − | <td> | + | |
| − | + | ||
| − | + | ||
</tr> | </tr> | ||
| − | + | <tr> | |
| − | + | <td><p style="white-space: nowrap;">Negative Colony 1 Triplicate 2</p></td> | |
| − | + | <td><p>9.84E+07 CFU/mL</p></td> | |
| − | + | ||
| − | <td> | + | |
| − | + | ||
| − | + | ||
</tr> | </tr> | ||
| − | + | <tr> | |
| − | + | <td><p style="white-space: nowrap;">Negative Colony 1 Triplicate 3</p></td> | |
| − | + | <td><p>1.6E+06 CFU/mL</p></td> | |
| − | + | ||
| − | <td> | + | |
| − | + | ||
| − | + | ||
</tr> | </tr> | ||
| − | + | <tr> | |
| − | + | <td><p style="white-space: nowrap;">Negative Colony 2 Triplicate 1</p></td> | |
| − | + | <td><p>1.376E+08 CFU/mL</p></td> | |
| − | + | ||
| − | <td> | + | |
| − | + | ||
| − | + | ||
</tr> | </tr> | ||
| − | + | <tr> | |
| − | + | <td><p style="white-space: nowrap;">Negative Colony 2 Triplicate 2</p></td> | |
| − | + | <td><p>1.008E+08 CFU/mL</p></td> | |
| − | + | </tr> | |
| − | <td> | + | <tr> |
| − | + | <td><p style="white-space: nowrap;">Negative Colony 2 Triplicate 1</p></td> | |
| − | + | <td><p>2.23E+07 CFU/mL</p></td> | |
| − | </tr>< | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | <td> | + | |
| − | + | ||
| − | + | ||
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</tbody> | </tbody> | ||
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Latest revision as of 13:46, 7 December 2018
Overview
Calibration 1 – LUDOX CL-X
Our first calibration uses LUDOX CL-X solution to convert absorbance (ABS600) to an equivalent measurement of optical density (OD 600). This is necessary because absorbance measurements are dependent on the depth of the fluid sample, while OD 600 values obtained from a standard spectrophotometer depend only on cuvette width, which is constant. This calibration eliminates measurement variations specific to our plate reader and returns standard values of optical density that other labs can reference.
Table 1: The data collected by LUDOX CL-X to calibrate OD600.
|
|
LUDOX CL-X |
H2O |
|---|---|---|
Replicate 1 |
0.085 |
0.042 |
Replicate 2 |
0.088 |
0.063 |
Replicate 3 |
0.086 |
0.046 |
Replicate 4 |
0.085 |
0.054 |
Arith. Mean |
0.086 |
0.051 |
Corrected Abs600 |
0.035 |
|
Reference OD600 |
0.063 |
|
OD600/Abs600 |
1.813 |
Calibration 2 – Microsphere Particle Standard Curve
For the next calibration, we prepared a dilution series of the silica microspheres found in the iGEM interlab kit. Because the concentration (particles per volume) is known, and because the microspheres have roughly the same size and optical qualities to bacterial cells, we can convert Abs600 values to number of cells.
Table 2: The data collected by silica beads to find the relationship between particles and Abs600.
|
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 |
4.6E+05 |
2.3E+05 |
0 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
Replicate 1 |
0.884 |
0.536 |
0.277 |
0.231 |
0.087 |
0.057 |
0.045 |
0.036 |
0.036 |
0.032 |
0.030 |
0.029 |
|
Replicate 2 |
0.897 |
0.430 |
0.223 |
0.133 |
0.077 |
0.056 |
0.043 |
0.036 |
0.033 |
0.033 |
0.029 |
0.030 |
|
Replicate 3 |
1.082 |
0.417 |
0.226 |
0.124 |
0.075 |
0.054 |
0.061 |
0.039 |
0.037 |
0.067 |
0.033 |
0.029 |
|
Replicate 4 |
1.484 |
0.596 |
0.26 |
0.213 |
0.089 |
0.057 |
0.041 |
0.037 |
0.033 |
0.032 |
0.03 |
0.031 |
|
Arith. Mean |
1.087 |
0.495 |
0.247 |
0.175 |
0.082 |
0.056 |
0.048 |
0.037 |
0.035 |
0.041 |
0.031 |
0.030 |
|
Arith. STDEV |
0.280 |
0.086 |
0.026 |
0.055 |
0.007 |
0.001 |
0.009 |
0.001 |
0.002 |
0.017 |
0.002 |
0.001 |
|
Arith. Net Mean |
1.057 |
0.465 |
0.217 |
0.146 |
0.052 |
0.026 |
0.018 |
0.007 |
0.005 |
0.011 |
0.001 |
|
Calibration 3 – Fluorescence Standard Curve
Our final calibration tackles the problem of inconsistencies in fluorescence measurements from model to model. In order to standardize our fluorescence values we create a standard fluorescence curve using the protein fluorescein. Measuring the fluorescence of a dilution series of fluorescein allows us to obtain a curve that describes the relationship between fluorescence values and fluorescein concentration.
Table 3: The data collected by fluorescein to find the relationship between fluorescence and fluorescein concentration.
|
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 |
148873 |
85179 |
41074 |
23229 |
12491 |
5683 |
2437 |
1237 |
426 |
180 |
84 |
25 |
|
Replicate 2 |
151337 |
79470 |
41795 |
20500 |
10628 |
5217 |
2720 |
1378 |
684 |
368 |
173 |
22 |
|
Replicate 3 |
150211 |
84298 |
44059 |
22273 |
11429 |
5658 |
2892 |
1430 |
715 |
357 |
179 |
25 |
|
Replicate 4 |
153849 |
84629 |
45004 |
23125 |
11754 |
5867 |
2827 |
1548 |
890 |
425 |
235 |
21 |
|
Arith. Mean |
1.511E+05 |
8.339E+04 |
4.298E+04 |
2.228E+04 |
1.158E+04 |
5.606E+03 |
2.719E+03 |
1.398E+03 |
6.788E+02 |
3.325E+02 |
1.678E+02 |
2.325E+01 |
|
Arith. STDEV |
2.110E+03 |
2.641E+03 |
1.853E+03 |
1.263E+03 |
7.723E+02 |
2.757E+02 |
2.009E+02 |
1.289E+02 |
1.914E+02 |
1.059E+02 |
6.243E+01 |
2.062E+00 |
|
Arith. Net Mean |
1.510E+05 |
8.337E+04 |
4.296E+04 |
2.226E+04 |
1.155E+04 |
5.583E+03 |
2.696E+03 |
1.375E+03 |
6.555E+02 |
3.093E+02 |
1.445E+02 |
|
Cell Measurement
Once our calibrations were complete, we moved on to the cell measurement protocol. First we transformed competent DH5-alpha with the 8 plasmids provided in the kit. After incubating for 16 hours at 37 degrees Celsius and 220 rpm, we diluted the solutions to an Abs600 value of 0.02 before measuring both OD 600 as well as fluorescence at 0 hours and 6 hours. Then, using our previous calibrations we converted fluorescence per OD to fluorescence per particle.
Table 4: Fluorescence Raw Readings in zero hour.
|
Hour 0: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Cr (blank) |
|---|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
906 |
1726 |
1167 |
777 |
1614 |
1026 |
1504 |
856 |
776 |
|
Colony 1, Replicate 2 |
792 |
1682 |
1285 |
827 |
1583 |
1328 |
1346 |
751 |
813 |
|
Colony 1, Replicate 3 |
886 |
1617 |
1403 |
910 |
1366 |
1141 |
763 |
766 |
762 |
|
Colony 1, Replicate 4 |
812 |
1515 |
1405 |
861 |
1525 |
1202 |
1220 |
794 |
789 |
|
Colony 2, Replicate 1 |
851 |
1661 |
1466 |
1012 |
1332 |
1035 |
1265 |
811 |
875 |
|
Colony 2, Replicate 2 |
773 |
1560 |
1569 |
1074 |
1296 |
1089 |
1192 |
771 |
922 |
|
Colony 2, Replicate 3 |
833 |
1590 |
1469 |
904 |
1333 |
1091 |
1295 |
768 |
862 |
|
Colony 2, Replicate 4 |
745 |
1695 |
1519 |
781 |
1278 |
812 |
1136 |
848 |
991 |
Table 5: Fluorescence Raw Readings in sixth hour.
|
Hour 6: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Cr (blank) |
|---|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
975 |
5090 |
1471 |
1690 |
8573 |
1503 |
2042 |
942 |
894 |
|
Colony 1, Replicate 2 |
803 |
4603 |
1440 |
1601 |
8282 |
1476 |
1920 |
932 |
928 |
|
Colony 1, Replicate 3 |
926 |
4978 |
1427 |
1609 |
8246 |
1395 |
1847 |
882 |
1001 |
|
Colony 1, Replicate 4 |
850 |
4993 |
1566 |
1642 |
8688 |
1384 |
2113 |
1106 |
925 |
|
Colony 2, Replicate 1 |
1042 |
5576 |
1602 |
1171 |
4104 |
1276 |
1674 |
1063 |
940 |
|
Colony 2, Replicate 2 |
968 |
5312 |
1510 |
1188 |
4243 |
1368 |
1498 |
1111 |
893 |
|
Colony 2, Replicate 3 |
958 |
4621 |
1601 |
1343 |
4184 |
1419 |
1741 |
1136 |
937 |
|
Colony 2, Replicate 4 |
954 |
4957 |
1579 |
1095 |
3781 |
1255 |
1720 |
1036 |
984 |
Table 6: Abs600 Raw Readings in zero hour.
|
Hour 0: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Cr (blank) |
|---|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
0.089 |
0.088 |
0.083 |
0.078 |
0.066 |
0.068 |
0.07 |
0.227 |
0.034 |
|
Colony 1, Replicate 2 |
0.088 |
0.083 |
0.082 |
0.078 |
0.067 |
0.068 |
0.064 |
0.234 |
0.034 |
|
Colony 1, Replicate 3 |
0.087 |
0.08 |
0.082 |
0.08 |
0.061 |
0.066 |
0.036 |
0.065 |
0.035 |
|
Colony 1, Replicate 4 |
0.085 |
0.081 |
0.081 |
0.077 |
0.062 |
0.061 |
0.045 |
0.068 |
0.034 |
|
Colony 2, Replicate 1 |
0.08 |
0.091 |
0.079 |
0.082 |
0.07 |
0.073 |
0.073 |
0.298 |
0.036 |
|
Colony 2, Replicate 2 |
0.086 |
0.081 |
0.075 |
0.087 |
0.067 |
0.071 |
0.201 |
0.297 |
0.035 |
|
Colony 2, Replicate 3 |
0.082 |
0.085 |
0.074 |
0.082 |
0.07 |
0.069 |
0.075 |
0.257 |
0.033 |
|
Colony 2, Replicate 4 |
0.086 |
0.086 |
0.078 |
0.083 |
0.07 |
0.068 |
0.07 |
0.098 |
0.034 |
Table 7: Abs600 Raw Readings in sixth hour.
|
Hour 6: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Cr (blank) |
|---|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
0.762 |
0.459 |
0.467 |
0.497 |
0.78 |
0.632 |
0.652 |
0.539 |
0.038 |
|
Colony 1, Replicate 2 |
0.644 |
0.44 |
0.46 |
0.544 |
0.751 |
0.615 |
0.642 |
0.576 |
0.035 |
|
Colony 1, Replicate 3 |
0.653 |
0.475 |
0.558 |
0.538 |
0.85 |
0.613 |
0.661 |
0.589 |
0.04 |
|
Colony 1, Replicate 4 |
0.676 |
0.586 |
0.561 |
0.576 |
0.803 |
0.654 |
0.664 |
0.528 |
0.045 |
|
Colony 2, Replicate 1 |
0.559 |
0.507 |
0.476 |
0.457 |
0.498 |
0.743 |
0.687 |
0.655 |
0.036 |
|
Colony 2, Replicate 2 |
0.686 |
0.514 |
0.453 |
0.458 |
0.524 |
0.693 |
0.799 |
0.62 |
0.041 |
|
Colony 2, Replicate 3 |
0.655 |
0.553 |
0.512 |
0.519 |
0.607 |
0.889 |
0.86 |
0.648 |
0.037 |
|
Colony 2, Replicate 4 |
0.66 |
0.601 |
0.463 |
0.541 |
0.575 |
0.819 |
0.779 |
0.67 |
0.035 |
Table 8: uM Fluorescein / OD in zero hour.
|
Hour 0: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
0.074 |
0.552 |
0.250 |
0.001 |
0.822 |
0.231 |
0.634 |
0.013 |
|
Colony 1, Replicate 2 |
-0.012 |
0.556 |
0.309 |
0.010 |
0.732 |
0.475 |
0.557 |
-0.010 |
|
Colony 1, Replicate 3 |
0.075 |
0.596 |
0.428 |
0.103 |
0.729 |
0.384 |
0.031 |
0.004 |
|
Colony 1, Replicate 4 |
0.014 |
0.485 |
0.411 |
0.053 |
0.825 |
0.480 |
1.229 |
0.005 |
|
Colony 2, Replicate 1 |
-0.017 |
0.448 |
0.431 |
0.093 |
0.422 |
0.136 |
0.331 |
-0.008 |
|
Colony 2, Replicate 2 |
-0.092 |
0.435 |
0.507 |
0.092 |
0.367 |
0.146 |
0.051 |
-0.018 |
|
Colony 2, Replicate 3 |
-0.019 |
0.439 |
0.464 |
0.027 |
0.399 |
0.200 |
0.323 |
-0.013 |
|
Colony 2, Replicate 4 |
-0.148 |
0.425 |
0.376 |
-0.134 |
0.250 |
-0.165 |
0.126 |
-0.070 |
Table 9: uM Fluorescein / OD in sixth hour
|
Hour 6: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
0.004 |
0.313 |
0.042 |
0.051 |
0.325 |
0.032 |
0.059 |
0.003 |
|
Colony 1, Replicate 2 |
-0.006 |
0.285 |
0.038 |
0.044 |
0.322 |
0.030 |
0.051 |
0.000 |
|
Colony 1, Replicate 3 |
-0.004 |
0.287 |
0.026 |
0.040 |
0.281 |
0.022 |
0.043 |
-0.007 |
|
Colony 1, Replicate 4 |
-0.004 |
0.236 |
0.039 |
0.049 |
0.321 |
0.024 |
0.060 |
0.012 |
|
Colony 2, Replicate 1 |
0.006 |
0.309 |
0.051 |
0.017 |
0.215 |
0.015 |
0.035 |
0.006 |
|
Colony 2, Replicate 2 |
0.004 |
0.293 |
0.044 |
0.022 |
0.218 |
0.023 |
0.025 |
0.012 |
|
Colony 2, Replicate 3 |
0.001 |
0.224 |
0.042 |
0.026 |
0.179 |
0.018 |
0.031 |
0.010 |
|
Colony 2, Replicate 4 |
-0.002 |
0.220 |
0.038 |
0.007 |
0.163 |
0.011 |
0.031 |
0.003 |
Table 10: Net Fluorescein a.u. in zero hour.
|
Hour 0: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
130.00 |
950.00 |
391.00 |
1.00 |
838.00 |
250.00 |
728.00 |
80.00 |
|
Colony 1, Replicate 2 |
-21.00 |
869.00 |
472.00 |
14.00 |
770.00 |
515.00 |
533.00 |
-62.00 |
|
Colony 1, Replicate 3 |
124.00 |
855.00 |
641.00 |
148.00 |
604.00 |
379.00 |
1.00 |
4.00 |
|
Colony 1, Replicate 4 |
23.00 |
726.00 |
616.00 |
72.00 |
736.00 |
413.00 |
431.00 |
5.00 |
|
Colony 2, Replicate 1 |
-24.00 |
786.00 |
591.00 |
137.00 |
457.00 |
160.00 |
390.00 |
-64.00 |
|
Colony 2, Replicate 2 |
-149.00 |
638.00 |
647.00 |
152.00 |
374.00 |
167.00 |
270.00 |
-151.00 |
|
Colony 2, Replicate 3 |
-29.00 |
728.00 |
607.00 |
42.00 |
471.00 |
229.00 |
433.00 |
-94.00 |
|
Colony 2, Replicate 4 |
-246.00 |
704.00 |
528.00 |
-210.00 |
287.00 |
-179.00 |
145.00 |
-143.00 |
Table 11: Net Fluorescein a.u. in sixth hour.
|
Hour 6: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
81.00 |
4196.00 |
577.00 |
708.00 |
7679.00 |
609.00 |
1148.00 |
48.00 |
|
Colony 1, Replicate 2 |
-125.00 |
3675.00 |
512.00 |
582.00 |
7354.00 |
548.00 |
992.00 |
4.00 |
|
Colony 1, Replicate 3 |
-75.00 |
3977.00 |
426.00 |
600.00 |
7245.00 |
394.00 |
846.00 |
-119.00 |
|
Colony 1, Replicate 4 |
-75.00 |
4068.00 |
641.00 |
654.00 |
7763.00 |
459.00 |
1188.00 |
181.00 |
|
Colony 2, Replicate 1 |
102.00 |
4636.00 |
750.00 |
231.00 |
3164.00 |
336.00 |
734.00 |
123.00 |
|
Colony 2, Replicate 2 |
75.00 |
4419.00 |
708.00 |
295.00 |
3350.00 |
475.00 |
605.00 |
218.00 |
|
Colony 2, Replicate 3 |
21.00 |
3684.00 |
672.00 |
406.00 |
3247.00 |
482.00 |
804.00 |
199.00 |
|
Colony 2, Replicate 4 |
-30.00 |
3973.00 |
658.00 |
111.00 |
2797.00 |
271.00 |
736.00 |
52.00 |
Table 12: Net Abs600 in zero hour.
|
Hour 0: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
0.055 |
0.054 |
0.049 |
0.044 |
0.032 |
0.034 |
0.036 |
0.193 |
|
Colony 1, Replicate 2 |
0.054 |
0.049 |
0.048 |
0.044 |
0.033 |
0.034 |
0.030 |
0.200 |
|
Colony 1, Replicate 3 |
0.052 |
0.045 |
0.047 |
0.045 |
0.026 |
0.031 |
0.001 |
0.030 |
|
Colony 1, Replicate 4 |
0.051 |
0.047 |
0.047 |
0.043 |
0.028 |
0.027 |
0.011 |
0.034 |
|
Colony 2, Replicate 1 |
0.044 |
0.055 |
0.043 |
0.046 |
0.034 |
0.037 |
0.037 |
0.262 |
|
Colony 2, Replicate 2 |
0.051 |
0.046 |
0.040 |
0.052 |
0.032 |
0.036 |
0.166 |
0.262 |
|
Colony 2, Replicate 3 |
0.049 |
0.052 |
0.041 |
0.049 |
0.037 |
0.036 |
0.042 |
0.224 |
|
Colony 2, Replicate 4 |
0.052 |
0.052 |
0.044 |
0.049 |
0.036 |
0.034 |
0.036 |
0.064 |
Table 13: Net Abs600 in sixth hour.
|
Hour 6: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
0.724 |
0.421 |
0.429 |
0.438 |
0.742 |
0.594 |
0.614 |
0.501 |
|
Colony 1, Replicate 2 |
0.609 |
0.405 |
0.425 |
0.418 |
0.716 |
0.580 |
0.607 |
0.541 |
|
Colony 1, Replicate 3 |
0.613 |
0.435 |
0.518 |
0.472 |
0.810 |
0.573 |
0.621 |
0.549 |
|
Colony 1, Replicate 4 |
0.631 |
0.541 |
0.516 |
0.418 |
0.758 |
0.609 |
0.619 |
0.483 |
|
Colony 2, Replicate 1 |
0.523 |
0.471 |
0.461 |
0.421 |
0.462 |
0.707 |
0.651 |
0.619 |
|
Colony 2, Replicate 2 |
0.645 |
0.473 |
0.503 |
0.417 |
0.483 |
0.652 |
0.758 |
0.579 |
|
Colony 2, Replicate 3 |
0.618 |
0.516 |
0.501 |
0.482 |
0.570 |
0.852 |
0.823 |
0.611 |
|
Colony 2, Replicate 4 |
0.625 |
0.566 |
0.541 |
0.506 |
0.540 |
0.784 |
0.744 |
0.635 |
Table 14: Molecules of equivalent fluorescein per particle in zero hour.
|
Hour 0: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
2.34E+04 |
9.45E+03 |
2.71E+01 |
2.53E+04 |
1.04E+04 |
2.85E+04 |
2.95E+03 |
0.00E+00 |
|
Colony 1, Replicate 2 |
2.36E+04 |
1.16E+04 |
3.88E+02 |
2.33E+04 |
2.08E+04 |
2.08E+04 |
-2.75E+03 |
0.00E+00 |
|
Colony 1, Replicate 3 |
2.45E+04 |
1.57E+04 |
3.42E+03 |
1.70E+04 |
1.80E+04 |
-1.12E+03 |
-3.06E+04 |
-1.20E+03 |
|
Colony 1, Replicate 4 |
1.81E+04 |
1.38E+04 |
-3.96E+02 |
2.01E+04 |
1.55E+04 |
1.70E+04 |
-9.79E+03 |
-3.36E+03 |
|
Colony 2, Replicate 1 |
1.79E+04 |
1.64E+04 |
2.78E+03 |
1.19E+04 |
4.42E+03 |
1.23E+04 |
-3.99E+03 |
-2.39E+02 |
|
Colony 2, Replicate 2 |
2.02E+04 |
1.84E+04 |
7.05E+03 |
1.11E+04 |
9.43E+03 |
1.22E+04 |
-7.29E+02 |
3.04E+02 |
|
Colony 2, Replicate 3 |
1.53E+04 |
1.30E+04 |
-2.82E+03 |
9.28E+03 |
3.59E+03 |
1.12E+04 |
-7.06E+03 |
-7.66E+02 |
|
Colony 2, Replicate 4 |
1.80E+04 |
1.35E+04 |
-6.35E+03 |
7.79E+03 |
-6.61E+03 |
5.67E+03 |
-5.28E+03 |
0.00E+00 |
Table 15: Molecules of equivalent fluorescein per particle in sixth hour.
|
Hour 6: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
1.49E+02 |
1.33E+04 |
1.79E+03 |
2.15E+03 |
1.38E+04 |
1.36E+03 |
2.49E+03 |
1.27E+02 |
|
Colony 1, Replicate 2 |
-2.73E+02 |
1.21E+04 |
1.60E+03 |
1.85E+03 |
1.37E+04 |
1.26E+03 |
2.17E+03 |
9.83E+00 |
|
Colony 1, Replicate 3 |
-1.63E+02 |
1.22E+04 |
1.09E+03 |
1.69E+03 |
1.19E+04 |
9.14E+02 |
1.81E+03 |
-2.88E+02 |
|
Colony 1, Replicate 4 |
-1.58E+02 |
1.00E+04 |
1.65E+03 |
2.08E+03 |
1.36E+04 |
1.00E+03 |
2.55E+03 |
4.98E+02 |
|
Colony 2, Replicate 1 |
2.59E+02 |
1.31E+04 |
2.16E+03 |
7.30E+02 |
9.11E+03 |
6.32E+02 |
1.50E+03 |
2.64E+02 |
|
Colony 2, Replicate 2 |
1.55E+02 |
1.24E+04 |
1.87E+03 |
9.41E+02 |
9.22E+03 |
9.69E+02 |
1.06E+03 |
5.01E+02 |
|
Colony 2, Replicate 3 |
4.52E+01 |
9.49E+03 |
1.78E+03 |
1.12E+03 |
7.57E+03 |
7.52E+02 |
1.30E+03 |
4.33E+02 |
|
Colony 2, Replicate 4 |
-6.38E+01 |
9.33E+03 |
1.62E+03 |
2.92E+02 |
6.89E+03 |
4.60E+02 |
1.32E+03 |
1.09E+02 |
Table 16: Net Fluorescein a.u. in zero hour.
|
Hour 0: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
950.00 |
391.00 |
1.00 |
838.00 |
250.00 |
728.00 |
80.00 |
0.00 |
|
Colony 1, Replicate 2 |
869.00 |
472.00 |
14.00 |
770.00 |
515.00 |
533.00 |
-62.00 |
0.00 |
|
Colony 1, Replicate 3 |
828.00 |
614.00 |
121.00 |
577.00 |
352.00 |
-26.00 |
-23.00 |
-27.00 |
|
Colony 1, Replicate 4 |
640.00 |
530.00 |
-14.00 |
650.00 |
327.00 |
345.00 |
-81.00 |
-86.00 |
|
Colony 2, Replicate 1 |
739.00 |
544.00 |
90.00 |
410.00 |
113.00 |
343.00 |
-111.00 |
-47.00 |
|
Colony 2, Replicate 2 |
698.00 |
707.00 |
212.00 |
434.00 |
227.00 |
330.00 |
-91.00 |
60.00 |
|
Colony 2, Replicate 3 |
599.00 |
478.00 |
-87.00 |
342.00 |
100.00 |
304.00 |
-223.00 |
-129.00 |
|
Colony 2, Replicate 4 |
704.00 |
528.00 |
-210.00 |
287.00 |
-179.00 |
145.00 |
-143.00 |
0.00 |
Table 17: Net Fluorescein a.u. in sixth hour.
|
Hour 6: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
81.00 |
4196.00 |
577.00 |
708.00 |
7679.00 |
609.00 |
1148.00 |
48.00 |
|
Colony 1, Replicate 2 |
-125.00 |
3675.00 |
512.00 |
582.00 |
7354.00 |
548.00 |
992.00 |
4.00 |
|
Colony 1, Replicate 3 |
-75.00 |
3977.00 |
426.00 |
600.00 |
7245.00 |
394.00 |
846.00 |
-119.00 |
|
Colony 1, Replicate 4 |
-75.00 |
4068.00 |
641.00 |
654.00 |
7763.00 |
459.00 |
1188.00 |
181.00 |
|
Colony 2, Replicate 1 |
102.00 |
4636.00 |
750.00 |
231.00 |
3164.00 |
336.00 |
734.00 |
123.00 |
|
Colony 2, Replicate 2 |
75.00 |
4419.00 |
708.00 |
295.00 |
3350.00 |
475.00 |
605.00 |
218.00 |
|
Colony 2, Replicate 3 |
21.00 |
3684.00 |
672.00 |
406.00 |
3247.00 |
482.00 |
804.00 |
199.00 |
|
Colony 2, Replicate 4 |
-30.00 |
3973.00 |
658.00 |
111.00 |
2797.00 |
271.00 |
736.00 |
52.00 |
Table 18: Net Abs600 in zero hour.
|
Hour 0: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
0.054 |
0.055 |
0.049 |
0.044 |
0.032 |
0.034 |
0.036 |
0.193 |
|
Colony 1, Replicate 2 |
0.049 |
0.054 |
0.048 |
0.044 |
0.033 |
0.034 |
0.030 |
0.200 |
|
Colony 1, Replicate 3 |
0.045 |
0.052 |
0.047 |
0.045 |
0.026 |
0.031 |
0.001 |
0.030 |
|
Colony 1, Replicate 4 |
0.047 |
0.051 |
0.047 |
0.043 |
0.028 |
0.027 |
0.011 |
0.034 |
|
Colony 2, Replicate 1 |
0.055 |
0.044 |
0.043 |
0.046 |
0.034 |
0.037 |
0.037 |
0.262 |
|
Colony 2, Replicate 2 |
0.046 |
0.051 |
0.040 |
0.052 |
0.032 |
0.036 |
0.166 |
0.262 |
|
Colony 2, Replicate 3 |
0.052 |
0.049 |
0.041 |
0.049 |
0.037 |
0.036 |
0.042 |
0.224 |
|
Colony 2, Replicate 4 |
0.052 |
0.052 |
0.044 |
0.049 |
0.036 |
0.034 |
0.036 |
0.064 |
Table 19: Net Abs600 in sixth hour.
|
Hour 6: |
N. Control |
P. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
|---|---|---|---|---|---|---|---|---|
|
Colony 1, Replicate 1 |
0.724 |
0.421 |
0.429 |
0.438 |
0.742 |
0.594 |
0.614 |
0.501 |
|
Colony 1, Replicate 2 |
0.609 |
0.405 |
0.425 |
0.418 |
0.716 |
0.580 |
0.607 |
0.541 |
|
Colony 1, Replicate 3 |
0.613 |
0.435 |
0.518 |
0.472 |
0.810 |
0.573 |
0.621 |
0.549 |
|
Colony 1, Replicate 4 |
0.631 |
0.541 |
0.516 |
0.418 |
0.758 |
0.609 |
0.619 |
0.483 |
|
Colony 2, Replicate 1 |
0.523 |
0.471 |
0.461 |
0.421 |
0.462 |
0.707 |
0.651 |
0.619 |
|
Colony 2, Replicate 2 |
0.645 |
0.473 |
0.503 |
0.417 |
0.483 |
0.652 |
0.758 |
0.579 |
|
Colony 2, Replicate 3 |
0.618 |
0.516 |
0.501 |
0.482 |
0.570 |
0.852 |
0.823 |
0.611 |
|
Colony 2, Replicate 4 |
0.625 |
0.566 |
0.541 |
0.506 |
0.540 |
0.784 |
0.744 |
0.635 |
Colony Formig Units
Finally, we test whether our cell measurement conversion was accurate. Because our conversions are based on the calibrations we performed using the silica beads, we observe actual colony forming units on plates and compare with our theoretical conversions. To do this, we incubated 2 samples of both positive and negative control overnight, then diluted 1 mL of the culture to an OD 600 value of 0.1 and prepared a series dilution. We then spread the final 3 dilutions of each sample onto plates and counted the CFUs. Because we started with an OD 600 value of 0.1, we know the OD values of the subsequent dilutions and can compare the resulting number of CFUs to our conversions from our cell measurement protocol.
Sample |
CFU Concerntration |
|---|---|
Positive Colony 1 Triplicate 1 |
1.8E+07 CFU/mL |
Positive Colony 1 Triplicate 2 |
2.56E+07 CFU/mL |
Positive Colony 1 Triplicate 3 |
5.6E+06 CFU/mL |
Positive Colony 2 Triplicate 1 |
7.44E+07 CFU/mL |
Positive Colony 2 Triplicate 2 |
1.04E+07 CFU/mL |
Positive Colony 2 Triplicate 3 |
2.4E+06 CFU/mL |
Negative Colony 1 Triplicate 1 |
2.23E+07 CFU/mL |
Negative Colony 1 Triplicate 2 |
9.84E+07 CFU/mL |
Negative Colony 1 Triplicate 3 |
1.6E+06 CFU/mL |
Negative Colony 2 Triplicate 1 |
1.376E+08 CFU/mL |
Negative Colony 2 Triplicate 2 |
1.008E+08 CFU/mL |
Negative Colony 2 Triplicate 1 |
2.23E+07 CFU/mL |
|
Positive/Negative |
Colony |
Triplicate |
Dilution |
Data |
|---|---|---|---|---|
|
Positive |
Colony 1 |
Triplicate 1 |
Dilution 3 |
19 |
|
Positive |
Colony 1 |
Triplicate 1 |
Dilution 4 |
23 |
|
Positive |
Colony 1 |
Triplicate 1 |
Dilution 5 |
0 |
|
Positive |
Colony 1 |
Triplicate 2 |
Dilution 3 |
44 |
|
Positive |
Colony 1 |
Triplicate 2 |
Dilution 4 |
32 |
|
Positive |
Colony 1 |
Triplicate 2 |
Dilution 5 |
0 |
|
Positive |
Colony 1 |
Triplicate 3 |
Dilution 3 |
173 |
|
Positive |
Colony 1 |
Triplicate 3 |
Dilution 4 |
7 |
|
Positive |
Colony 1 |
Triplicate 3 |
Dilution 5 |
0 |
|
Positive |
Colony 2 |
Triplicate 1 |
Dilution 3 |
20 |
|
Positive |
Colony 2 |
Triplicate 1 |
Dilution 4 |
93 |
|
Positive |
Colony 2 |
Triplicate 1 |
Dilution 5 |
2 |
|
Positive |
Colony 2 |
Triplicate 2 |
Dilution 3 |
100 |
|
Positive |
Colony 2 |
Triplicate 2 |
Dilution 4 |
13 |
|
Positive |
Colony 2 |
Triplicate 2 |
Dilution 5 |
3 |
|
Positive |
Colony 2 |
Triplicate 3 |
Dilution 3 |
3 |
|
Positive |
Colony 2 |
Triplicate 3 |
Dilution 4 |
0 |
|
Positive |
Colony 2 |
Triplicate 3 |
Dilution 5 |
0 |
|
Negative |
Colony 1 |
Triplicate 1 |
Dilution 3 |
13 |
|
Negative |
Colony 1 |
Triplicate 1 |
Dilution 4 |
29 |
|
Negative |
Colony 1 |
Triplicate 1 |
Dilution 5 |
1368 |
|
Negative |
Colony 1 |
Triplicate 2 |
Dilution 3 |
355 |
|
Negative |
Colony 1 |
Triplicate 2 |
Dilution 4 |
123 |
|
Negative |
Colony 1 |
Triplicate 2 |
Dilution 5 |
2 |
|
Negative |
Colony 1 |
Triplicate 3 |
Dilution 3 |
431 |
|
Negative |
Colony 1 |
Triplicate 3 |
Dilution 4 |
2 |
|
Negative |
Colony 1 |
Triplicate 3 |
Dilution 5 |
1 |
|
Negative |
Colony 2 |
Triplicate 1 |
Dilution 3 |
88 |
|
Negative |
Colony 2 |
Triplicate 1 |
Dilution 4 |
172 |
|
Negative |
Colony 2 |
Triplicate 1 |
Dilution 5 |
3 |
|
Negative |
Colony 2 |
Triplicate 2 |
Dilution 3 |
370 |
|
Negative |
Colony 2 |
Triplicate 2 |
Dilution 4 |
26 |
|
Negative |
Colony 2 |
Triplicate 2 |
Dilution 5 |
1 |
|
Negative |
Colony 2 |
Triplicate 3 |
Dilution 3 |
275 |
|
Negative |
Colony 2 |
Triplicate 3 |
Dilution 4 |
29 |
|
Negative |
Colony 2 |
Triplicate 3 |
Dilution 5 |
0 |
Extra credit – Flow Cytometry
As an extra credit task, we also collected flow cytometry data using an ACEA NovoCyte Flow Cytometer as well as SpheroTech Rainbow Calibration Particles model URCP-38-2K. Flow cytometry provides an even more accurate method of counting particles.
Conclusion
We hope our experimentation will help to improve the accessibility and comparability of reliable data in synthetic biology labs across the world. Thank you to iGEM for providing this opportunity to give back to the scientific community.
