Team:NUDT CHINA/InterLab
InterLab
Introduction
The aim of Interlab is to establish a comparable “quantitative expression” of the key components measurement in labs around the world. In order to contribute to the overall goal, our team followed the Interlab protocol to conduct experiments and record results. Through the experiments, we were able to get the OD600 and Fluorescence measurements of E.coli strain DH5α as well as quotient between the two in different time period. In this way, we are providing a source of data for Interlab project to analyze and recruit into its broader picture.
Methods and Design
1. InterLab Parts
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_J364003):
Test Device 5 (BBa_J364004):
Test Device 6 (BBa_J364005):
2. Preparation
To start with, our team transformed E.coli strain DH5α with the provided plasmids, namely Test Device 1,2,3,4,5,6, Positive Control, and Negative Control. As long as colonies had emerged on Cm+ Resistance Media, we picked up 2 colonies in each petrie dish and cultured them at 37℃ with 220 rpm frequency for 14 hours. When the bacteria solutions were turbid enough, we began the following process.
3. OD600 Reference Point
With plate reader, we measured Abs600 of the LUDOX and H2O. The H2O measurement served as the background. Both included 4 technical replicates to enhance the reliability of the results. Comparing to the standard OD600 reference given, which was 0.0425, we were able to achieve a ratio between OD600 and Abs600. The ratio was essential in converting Abs600 raw measurements into standard OD600 records.
4. Fluorescein Standard Curve
Different concentrations of fluorescein were obtained by 2-fold serial dilution. In the first pipet, 150μL PBS and 50μL 2×Fluorescein were added. By removing half of the 200μL solution in previous pipets to later ones which already contained 100μL PBS, we were able to generate 11 solutions in half-descending fluorescence concentration. We also included another pipet with 100μL PBS only as blank. Within each concentration, we performed 4 replicates to calculate the more representative mean. After the results were recorded, all fluorescein concentrations were divided by average fluorescence measurement, of which the medium-high mean were calculated to diminish operation errors. This mean point would be used to set up the conversion between fluorescein concentration and fluorescence measurements in later steps.
5. OD600 and Fluorescence Measurements
We tracked the original OD600 of the 16 samples so as to dilute them into 0.02. Once the original values were available, we integrated them into the dilution calculation sheet and followed the suggested volume to dilute each sample. After dilution, we confirmed that OD600 had reached exactly 0.02 or around. Our team set that time as T=0(h) and measured average OD600 from 4 replicates of each 16 samples to reduce technical error. Other 4 replicates were used for T=0(h) fluorescence measurements, with the same equipment and settings in Step 3. Henceforward, we recorded the OD600 and fluorescence every 2 hours, when T=2, T=4, and T=6.
6. Equipments and Settings
To obtain OD600 measurement, we employed Multiscan FC. Single wavelength of 600nm was used, and path length correction was turned off. To reduce measurement error, we also added a dynamic circulation of 5 times, with 2-second intervals.
The fluorescence measurement was obtained through Fluoroskan Ascent FL and Thermo Fisher. The filters used were 485nm and 538nm for excitation and emission respectively. The measurement cycled 5 times with intervals of 2 second.
Results & Discussion
1. Calibration
First of all, after 4 technical replicates of measuring Abs600 of LUDOX-HS40 and H2O, we got a specific value, 5.12.
|
|
LUDOX-HS40 |
H2O |
|
Replicate 1 |
0.0461 |
0.0392 |
|
Replicate 2 |
0.0475 |
0.0389 |
|
Replicate 3 |
0.0472 |
0.0392 |
|
Replicate 4 |
0.0471 |
0.0374 |
|
Arith. Mean |
0.046975 |
0.038675 |
|
Corrected Abs600 |
0.0083 |
|
|
Reference OD600 |
0.0425 |
|
|
OD600/Abs600 |
5.12048193 |
|
Fig.1 OD600 reference point
Secondly, following the protocol, we prepared a dilution series of fluorescein from 50μM fluorescein concentration to 0.05μM fluorescein concentration in 4 replicates, double diluted each time. Then we got a fluorescein fluorescence standard curve, which is shown as below.
Fig.2 Fluorescein standard curve
Besides, we also need a mean μM Fluorescein data. Because medium-high points likely to be less impacted by saturation or pipetting error, we chose data from 25μM to 1.56μM to calculate the ratio between concentration and fluorescence. 0.0096424 is the result.
2.Cell measurement
2.1 Abs600
We have done measurements with 2 biological replicates and 4 technical replicates, and results are basically the same.
When we used colony 1 group, result shows that as time went by, Abs600 values of all devices increase one by one. Abs600 values for device 3 are almost the highest at each check point while device 1 are almost the lowest. And a situation worthy of attention is that Abs600 values of negative control at all point are higher than the counterparts of positive control.
Fig.3 Abs600-Time curves (Colony 1)
|
Abs600 Raw Readings: |
|||||
|
|
|
Hour 0 |
Hour 2 |
Hour 4 |
Hour 6 |
|
Neg. Control |
Replicate1 |
0.247831 |
0.395813 |
1.213554 |
1.743627 |
|
Replicate2 |
0.252952 |
0.428072 |
1.219699 |
1.887512 |
|
|
Replicate3 |
0.245271 |
0.44753 |
1.220211 |
1.842145 |
|
|
Replicate4 |
0.249367 |
0.459307 |
1.137259 |
1.909325 |
|
|
Pos. Control |
Replicate1 |
0.27753 |
0.331807 |
0.908373 |
1.452271 |
|
Replicate2 |
0.250904 |
0.344608 |
0.850512 |
1.494361 |
|
|
Replicate3 |
0.273434 |
0.34512 |
0.920663 |
1.523958 |
|
|
Replicate4 |
0.245783 |
0.347681 |
0.829006 |
1.538193 |
|
|
Device 1 |
Replicate1 |
0.264217 |
0.364066 |
0.799307 |
1.278892 |
|
Replicate2 |
0.261657 |
0.358946 |
0.771145 |
1.322518 |
|
|
Replicate3 |
0.26012 |
0.340512 |
0.724548 |
1.375464 |
|
|
Replicate4 |
0.259096 |
0.347681 |
0.765512 |
1.321289 |
|
|
Device 2 |
Replicate1 |
0.245271 |
0.400422 |
1.045602 |
1.605169 |
|
Replicate2 |
0.249367 |
0.40503 |
1.101416 |
1.625548 |
|
|
Replicate3 |
0.244247 |
0.377892 |
1.038946 |
1.595337 |
|
|
Replicate4 |
0.251928 |
0.376867 |
0.989789 |
1.590319 |
|
|
Device 3 |
Replicate1 |
0.250904 |
0.496175 |
1.309819 |
1.852078 |
|
Replicate2 |
0.257048 |
0.516657 |
1.268855 |
1.951928 |
|
|
Replicate3 |
0.249367 |
0.437289 |
1.263735 |
1.958789 |
|
|
Replicate4 |
0.247831 |
0.409127 |
1.25247 |
1.968723 |
|
|
Device 4 |
Replicate1 |
0.253464 |
0.332831 |
0.857681 |
1.477566 |
|
Replicate2 |
0.248343 |
0.33744 |
0.819789 |
1.568916 |
|
|
Replicate3 |
0.244759 |
0.339488 |
0.805964 |
1.530307 |
|
|
Replicate4 |
0.241175 |
0.329759 |
0.782922 |
1.54659 |
|
|
Device 5 |
Replicate1 |
0.242711 |
0.381476 |
1.020512 |
1.722837 |
|
Replicate2 |
0.244247 |
0.3825 |
1.052259 |
1.715669 |
|
|
Replicate3 |
0.242199 |
0.38506 |
1.068645 |
1.74291 |
|
|
Replicate4 |
0.250392 |
0.36509 |
0.944729 |
1.704096 |
|
|
Device 6 |
Replicate1 |
0.246295 |
0.536627 |
1.310843 |
1.762572 |
|
Replicate2 |
0.248855 |
0.500271 |
1.313916 |
1.842657 |
|
|
Replicate3 |
0.250392 |
0.429608 |
1.299578 |
1.795036 |
|
|
Replicate4 |
0.259608 |
0.413735 |
1.288313 |
1.76206 |
|
|
LB + Chlor (blank) |
Replicate1 |
0.226837 |
0.228886 |
0.238102 |
0.230627 |
|
Replicate2 |
0.231958 |
0.230422 |
0.239127 |
0.231855 |
|
|
Replicate3 |
0.233494 |
0.230422 |
0.237078 |
0.232367 |
|
|
Replicate4 |
0.230934 |
0.227349 |
0.236054 |
0.230422 |
|
Fig.4 Raw Abs600 values (Colony 1)
Then we used colony 2 series to do same experiments. The overall trend is in agreement to colony 1 group. However, Abs600 value of device 6 and negative control exceed device 3 to be the first and second highest. Even though Positive control had the highest value at the very beginning, it is still the lowest one at each check point during the following 6 hours.
Fig.5 Abs600-Time curves (Colony 2)
|
Abs600 Raw Readings: |
|||||
|
|
|
Hour 0 |
Hour 2 |
Hour 4 |
Hour 6 |
|
Neg. Control |
Replicate1 |
0.250904 |
0.445482 |
1.272952 |
1.885157 |
|
Replicate2 |
0.249367 |
0.454699 |
1.278072 |
1.968416 |
|
|
Replicate3 |
0.252952 |
0.451114 |
1.210482 |
1.957663 |
|
|
Replicate4 |
0.266265 |
0.479789 |
1.179247 |
1.888741 |
|
|
Pos. Control |
Replicate1 |
0.252952 |
0.441386 |
1.094759 |
1.654325 |
|
Replicate2 |
0.260633 |
0.406566 |
1.089127 |
1.751819 |
|
|
Replicate3 |
0.263193 |
0.417319 |
1.06762 |
1.733181 |
|
|
Replicate4 |
0.255512 |
0.411687 |
1.043554 |
1.656066 |
|
|
Device 1 |
Replicate1 |
0.325151 |
0.338976 |
0.372771 |
0.435548 |
|
Replicate2 |
0.321054 |
0.333855 |
0.370723 |
0.443946 |
|
|
Replicate3 |
0.324639 |
0.312349 |
0.361506 |
0.436982 |
|
|
Replicate4 |
0.312349 |
0.313373 |
0.360482 |
0.431145 |
|
|
Device 2 |
Replicate1 |
0.248855 |
0.363042 |
0.99491 |
1.513512 |
|
Replicate2 |
0.250904 |
0.369699 |
0.984157 |
1.578849 |
|
|
Replicate3 |
0.247831 |
0.352289 |
0.91503 |
1.591343 |
|
|
Replicate4 |
0.25756 |
0.346657 |
0.877139 |
1.56697 |
|
|
Device 3 |
Replicate1 |
0.27497 |
0.431657 |
1.202289 |
1.865289 |
|
Replicate2 |
0.256536 |
0.458795 |
1.25759 |
1.931855 |
|
|
Replicate3 |
0.25756 |
0.438825 |
1.224819 |
1.98388 |
|
|
Replicate4 |
0.25756 |
0.447018 |
1.121898 |
1.982139 |
|
|
Device 4 |
Replicate1 |
0.250392 |
0.313886 |
0.696898 |
1.278584 |
|
Replicate2 |
0.24988 |
0.310301 |
0.634428 |
1.281145 |
|
|
Replicate3 |
0.257048 |
0.308765 |
0.628795 |
1.278072 |
|
|
Replicate4 |
0.259096 |
0.290331 |
0.643645 |
1.216627 |
|
|
Device 5 |
Replicate1 |
0.246295 |
0.455723 |
1.186928 |
1.880241 |
|
Replicate2 |
0.25756 |
0.459307 |
1.204849 |
1.863651 |
|
|
Replicate3 |
0.246807 |
0.397349 |
1.103464 |
1.870614 |
|
|
Replicate4 |
0.263193 |
0.441386 |
1.141867 |
1.819512 |
|
|
Device 6 |
Replicate1 |
0.248855 |
0.487982 |
1.32006 |
1.972512 |
|
Replicate2 |
0.251416 |
0.483886 |
1.329789 |
1.963705 |
|
|
Replicate3 |
0.25244 |
0.451627 |
1.282681 |
1.934108 |
|
|
Replicate4 |
0.266777 |
0.459307 |
1.287289 |
1.937488 |
|
|
LB + Chlor (blank) |
Replicate1 |
0.238614 |
0.238102 |
0.23503 |
0.233904 |
|
Replicate2 |
0.23503 |
0.233494 |
0.236566 |
0.235645 |
|
|
Replicate3 |
0.236054 |
0.231446 |
0.236566 |
0.235952 |
|
|
Replicate4 |
0.241687 |
0.23247 |
0.245783 |
0.232265 |
|
Fig.6 Raw Abs600 values (Colony 2)
2.2 Fluorescence
We used same colonies to measure their fluorescence values.
As it’s shown in figure 7, all devices showed a trend of gradual increase in fluorescence overtime, with the highest absorbencies measured at the six hour time period. Fluorescence value of Device 1 increases consistently with the most dramatic speed. On the contrary, counterpart of negative control is always the lowest, not even the value, but also the rising velocity, and device 3 and 6 are only slightly higher than it.
Fig.7 Fluorescence-Time curves (Colony 1)
|
Fluorescence Raw Readings: |
|||||
|
|
|
Hour 0 |
Hour 2 |
Hour 4 |
Hour 6 |
|
Neg. Control |
Replicate1 |
5.209989 |
5.54977 |
6.472891 |
6.56813 |
|
Replicate2 |
5.171079 |
5.833241 |
6.529608 |
6.567257 |
|
|
Replicate3 |
5.186898 |
5.441483 |
6.383715 |
6.509024 |
|
|
Replicate4 |
5.194274 |
5.723555 |
6.463368 |
6.489255 |
|
|
Pos. Control |
Replicate1 |
5.445713 |
11.98757 |
33.11475 |
57.60393 |
|
Replicate2 |
5.631754 |
12.09197 |
33.24118 |
59.20011 |
|
|
Replicate3 |
5.729861 |
12.07664 |
33.56001 |
56.27913 |
|
|
Replicate4 |
5.592658 |
12.29495 |
32.67889 |
57.03549 |
|
|
Device 1 |
Replicate1 |
6.122431 |
16.42982 |
46.32978 |
96.19644 |
|
Replicate2 |
6.08099 |
16.28489 |
48.60549 |
97.23445 |
|
|
Replicate3 |
6.045781 |
16.54069 |
47.41807 |
94.82616 |
|
|
Replicate4 |
6.103347 |
16.39764 |
46.57271 |
97.67311 |
|
|
Device 2 |
Replicate1 |
6.826715 |
11.93965 |
36.66071 |
63.44618 |
|
Replicate2 |
6.897445 |
11.58005 |
36.30757 |
64.44318 |
|
|
Replicate3 |
6.819571 |
11.58714 |
36.80197 |
62.63553 |
|
|
Replicate4 |
6.884618 |
11.72881 |
36.34775 |
62.67168 |
|
|
Device 3 |
Replicate1 |
5.370117 |
5.788785 |
6.950749 |
7.478387 |
|
Replicate2 |
5.19102 |
5.815279 |
7.248347 |
7.62655 |
|
|
Replicate3 |
5.327616 |
5.767361 |
6.903009 |
7.339573 |
|
|
Replicate4 |
5.26142 |
5.70956 |
6.850868 |
7.419129 |
|
|
Device 4 |
Replicate1 |
5.251092 |
7.674111 |
23.33626 |
41.46638 |
|
Replicate2 |
5.247093 |
9.107355 |
23.64567 |
40.99201 |
|
|
Replicate3 |
5.195829 |
6.55446 |
22.55987 |
41.13167 |
|
|
Replicate4 |
5.268844 |
7.041709 |
23.45185 |
41.59823 |
|
|
Device 5 |
Replicate1 |
5.436595 |
6.638823 |
12.24985 |
14.22084 |
|
Replicate2 |
5.239953 |
6.462304 |
12.12886 |
13.7509 |
|
|
Replicate3 |
5.362731 |
6.459424 |
12.01427 |
14.17257 |
|
|
Replicate4 |
5.33499 |
6.362791 |
11.92326 |
14.05965 |
|
|
Device 6 |
Replicate1 |
5.150368 |
5.642443 |
6.511028 |
6.833299 |
|
Replicate2 |
5.171746 |
5.702248 |
6.532471 |
6.836482 |
|
|
Replicate3 |
5.181851 |
5.578135 |
6.600966 |
6.661619 |
|
|
Replicate4 |
5.206747 |
5.536729 |
6.481353 |
6.665059 |
|
|
LB + Chlor (blank) |
Replicate1 |
5.030219 |
5.069089 |
5.014596 |
5.051529 |
|
Replicate2 |
5.038815 |
5.019348 |
5.089624 |
5.0878 |
|
|
Replicate3 |
5.068299 |
5.072437 |
5.022567 |
5.031218 |
|
|
Replicate4 |
5.024612 |
5.02281 |
5.007114 |
5.072344 |
|
Fig.8 Raw Fluorescence values (Colony 1)
At the second time, results of almost all devices are in agreement to the first time, nevertheless, fluorescence value for device 1 is pretty different, which going down below device 2 and positive control from the 3rd hour, even device 4 also surpasses it at the 6th hour.
Fig.9 Fluorescence-Time curves (Colony 2)
|
Fluorescence Raw Readings: |
|||||
|
|
|
Hour 0 |
Hour 2 |
Hour 4 |
Hour 6 |
|
Neg. Control |
Replicate1 |
5.202063 |
5.409551 |
6.267453 |
6.546047 |
|
Replicate2 |
5.200951 |
5.388425 |
6.367106 |
6.457578 |
|
|
Replicate3 |
5.231905 |
5.459509 |
6.304455 |
6.376216 |
|
|
Replicate4 |
5.234977 |
5.444965 |
6.216507 |
6.388605 |
|
|
Pos. Control |
Replicate1 |
5.568839 |
11.24672 |
29.06219 |
50.52142 |
|
Replicate2 |
5.675913 |
11.23065 |
29.40039 |
50.62966 |
|
|
Replicate3 |
5.587093 |
11.24845 |
29.76562 |
50.56315 |
|
|
Replicate4 |
5.641905 |
11.59007 |
29.90887 |
51.81771 |
|
|
Device 1 |
Replicate1 |
7.212901 |
16.74134 |
22.27062 |
22.7243 |
|
Replicate2 |
7.188345 |
16.44796 |
22.47317 |
22.79647 |
|
|
Replicate3 |
7.201028 |
16.68378 |
23.05046 |
22.50906 |
|
|
Replicate4 |
7.214607 |
16.58921 |
22.72029 |
22.35301 |
|
|
Device 2 |
Replicate1 |
6.051227 |
11.31953 |
29.55128 |
58.52493 |
|
Replicate2 |
6.034464 |
11.29922 |
29.61808 |
60.38214 |
|
|
Replicate3 |
5.904204 |
10.94907 |
29.74428 |
59.57055 |
|
|
Replicate4 |
5.920397 |
11.38785 |
29.87139 |
59.63158 |
|
|
Device 3 |
Replicate1 |
5.225381 |
6.820621 |
6.643686 |
7.311257 |
|
Replicate2 |
5.270073 |
5.664928 |
6.787338 |
7.268843 |
|
|
Replicate3 |
5.207889 |
5.651378 |
6.671313 |
7.222882 |
|
|
Replicate4 |
5.222157 |
5.527013 |
6.700565 |
7.285775 |
|
|
Device 4 |
Replicate1 |
5.748372 |
8.142249 |
20.23301 |
33.43776 |
|
Replicate2 |
5.53358 |
8.084868 |
20.53106 |
31.92987 |
|
|
Replicate3 |
5.625829 |
6.961427 |
20.4742 |
32.44981 |
|
|
Replicate4 |
5.720001 |
7.355122 |
20.05921 |
32.24723 |
|
|
Device 5 |
Replicate1 |
5.545545 |
7.994489 |
11.85382 |
15.16459 |
|
Replicate2 |
5.469834 |
8.045887 |
11.73791 |
15.76424 |
|
|
Replicate3 |
5.406922 |
7.774544 |
12.08614 |
15.25091 |
|
|
Replicate4 |
5.410715 |
7.795197 |
11.5359 |
15.43117 |
|
|
Device 6 |
Replicate1 |
5.247958 |
5.53954 |
6.483918 |
6.671105 |
|
Replicate2 |
5.207919 |
5.557367 |
6.517664 |
6.478746 |
|
|
Replicate3 |
5.088215 |
5.498304 |
6.42272 |
6.447031 |
|
|
Replicate4 |
5.155286 |
5.35573 |
6.280222 |
6.586543 |
|
|
LB + Chlor (blank) |
Replicate1 |
5.033813 |
5.005738 |
5.056224 |
5.083954 |
|
Replicate2 |
5.032788 |
5.007401 |
5.068331 |
5.010431 |
|
|
Replicate3 |
5.071645 |
4.987419 |
5.072987 |
4.928791 |
|
|
Replicate4 |
5.053847 |
5.090735 |
5.018541 |
5.10907 |
|
Fig.10 Raw Fluorescence values (Colony 2)
2.3 Ratio between μM Fluorescein and OD600
We can’t tell which test device contains the stronger promoter only by judging their fluorescence values, so we normalized fluorescence values.
Fig.11 μM Fluorescein / OD600 curves
We also used negative natural logarithm to calculate.
Fig.12 -Ln (μM FITC / OD600) curve
The measurements of ratio between μM fluorescein and OD600 seem to be complex. There is obvious growth between 0h and 2h in positive control, device 1 and device 4. Then, the ratio started decreasing. Others show opposite trend during first two hours, but follow the downtrend up to hour 6. Eventually bacteria reach the stationary phase.
μM Fluorescein / OD600 also shows which test device contains the stronger promoter. Promoter strength is in the order of device 1, device 2, device 4, device 5, device 3 and device 6, with device 1 being the strongest. The constitutively expressed GFP in the positive control shows that that promoter has a strength in between device 2 and device 4. As expected, the negative control shows little to no μM Fluorescein / OD600.
