Team:NUDT CHINA/InterLab
Designed Protein Degradation Method Based on
Trim21 And Nanobody -- InterLab
InterLab
Introduction
The InterLab this year has been updated to a more detailed protocol. During the InterLab study, we used the LUDOX, fluorescence and the plasmids that were shipped along with the distribution kit, and closely followed the InterLab study protocol.We are really happy to receive the mail that our interlab study has beed accepted,it feels like a tiny victory!!
Through the InterLab 2018 study, we experienced a lot in the measuring work, and surprisingly got to know more about the instruments that usually were ignored by us. Actually, it is the measuring instruments that help us complete the project every year!!
Methods and Design
1. InterLab Parts
Positive Control (BBa_I20270)
NegativeControl (BBa_R0040)
Test Device1 (BBa_J364000)
Test Device2 (BBa_J364001)
Test Device3 (BBa_J364002)
Test Device4 (BBa_J364007)
Test Device5 (BBa_J364008)
Test Device6 (BBa_J364009)
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, 200μL Fluorescein 1×stock solution was 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 when T=0 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
Calibration
Calibration1: OD600 Reference point – LUDOX Protocol
We used LUDOX CL-X (45% colloidal silica suspension) , ddH2O and 96 well plate(black with clear flat bottom preferred) to get the conversion factor, which can transform Abs600 measurements into comparable OD600 measurements.
| LUDOX CL-X | H2O | |
|---|---|---|
| Replicate 1 |
0.062 |
0.039 |
| Replicate 2 |
0.060 |
0.039 |
| Replicate 3 |
0.066 |
0.038 |
| Replicate 4 |
0.061 |
0.038 |
| Arith. Mean |
0.062 |
0.039 |
| Corrected Abs600 |
0.024 | |
| Reference OD600 |
0.063 | |
| OD600/Abs600 |
2.654 |
Fig.1 OD600 reference point
Calibration2: Particle Standard Curve – Microsphere Protocol
Following the protocol, we prepared a dilution series of monodisperse silica microspheres and measure the Abs600 in our plate reader. We got a particle standard curve and transferred it into log scale, which are shown as below.
Fig.2 Particle standard curve
Fig.3 Particle standard curve (log scale)
The values of the Particle standard curve are provides for convenience in a table.
| 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 |
0.668 |
0.393 |
0.201 |
0.122 |
0.074 |
0.056 |
0.046 |
0.043 |
0.038 |
0.037 |
0.037 |
0.036 |
| Replicate 2 |
0.645 |
0.373 |
0.210 |
0.130 |
0.084 |
0.061 |
0.048 |
0.043 |
0.040 |
0.040 |
0.039 |
0.037 |
| Replicate 3 |
0.619 |
0.349 |
0.211 |
0.129 |
0.084 |
0.060 |
0.049 |
0.044 |
0.038 |
0.037 |
0.037 |
0.037 |
| Replicate 4 |
0.668 |
0.400 |
0.229 |
0.143 |
0.085 |
0.063 |
0.050 |
0.047 |
0.041 |
0.039 |
0.037 |
0.039 |
| Arith.Mean |
0.650 |
0.379 |
0.213 |
0.131 |
0.082 |
0.060 |
0.048 |
0.044 |
0.039 |
0.038 |
0.038 |
0.037 |
| Arith.Std.Dev. |
0.023 |
0.023 |
0.012 |
0.009 |
0.005 |
0.003 |
0.002 |
0.002 |
0.002 |
0.002 |
0.001 |
0.001 |
| Arith.Net Mean |
0.613 |
0.342 |
0.176 |
0.094 |
0.045 |
0.023 |
0.011 |
0.007 |
0.002 |
0.001 |
0.000 |
Fig.4 The values of Particle standard curve
Calibration3: Fluorescence standard curve – Fluorescein Protocol
We did a dilution series of fluorescein in four replicates. After recording the measurements, we generated a standard curve of fluorescence for fluorescein concentration and its log scale version, which are shown as below.
Fig.5 Particle standard curve
Fig.6 Particle standard curve (log scale)
We attached the details of the curves for reference.
| Fluorescein/a.u. | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Fluorescein uM | 10.00 | 5.00 | 2.50 | 1.25 | 0.63 | 0.31 | 0.16 | 0.08 | 0.04 | 0.02 | 0.01 |
| uM fluorescein/a.u.: | 4.38E-01 | 3.69E-01 | 3.50E-01 | 3.35E-01 | 3.29E-01 | 3.37E-01 | 3.31E-01 | 3.39E-01 | 3.37E-01 | 3.51E-01 | 3.97E-01 |
| Mean uM fluorescein/a.u.: | 3.44E-01 | ||||||||||
| MEFL/a.u.: | 2.07E+12 |
Fig.7 Detailed values of Fluorescein curve
Cell measurement
2.1 Abs600
We have read two plates at the time point: 0 and 6 hours with the same conditions in our calibration measurements.
We measured both fluorescence and absorbance of 2 biological replicates and 4 technical replicates. Abs600 values for device 5 are almost the lowest while the values of device 3 are almost the highest.
| Hour 0 | Hour 6 | ||
|---|---|---|---|
| Neg. Control | Replicate 1 | 0.068 | 0.297 |
| Replicate 2 | 0.069 | 0.285 | |
| Replicate 3 | 0.068 | 0.291 | Replicate 4 | 0.074 | 0.298 |
| Pos. Control | Replicate 1 | 0.072 | 0.296 | Replicate 2 | 0.071 | 0.288 | Replicate 3 | 0.075 | 0.284 | Replicate 4 | 0.068 | 0.285 |
| Device1 | Replicate 1 | 0.068 | 0.239 | Replicate 2 | 0.067 | 0.22 | Replicate 3 | 0.073 | 0.223 | Replicate 4 | 0.069 | 0.224 |
| Device2 | Replicate 1 | 0.066 | 0.268 | Replicate 2 | 0.071 | 0.27 | Replicate 3 | 0.068 | 0.268 | Replicate 4 | 0.066 | 0.273 |
| Device3 | Replicate 1 | 0.069 | 0.425 | Replicate 2 | 0.067 | 0.358 | Replicate 3 | 0.072 | 0.343 | Replicate 4 | 0.074 | 0.347 |
| Device4 | Replicate 1 | 0.067 | 0.21 | Replicate 2 | 0.067 | 0.217 | Replicate 3 | 0.067 | 0.217 | Replicate 4 | 0.07 | 0.221 |
| Device5 | Replicate 1 | 0.066 | 0.161 | Replicate 2 | 0.067 | 0.155 | Replicate 3 | 0.069 | 0.16 | Replicate 4 | 0.066 | 0.164 |
| Device6 | Replicate 1 | 0.067 | 0.294 | Replicate 2 | 0.068 | 0.284 | Replicate 3 | 0.067 | 0.286 | Replicate 4 | 0.071 | 0.285 |
| LB+Chior(blank) | Replicate 1 | 0.048 | 0.044 | Replicate 2 | 0.048 | 0.046 | Replicate 3 | 0.047 | 0.046 | Replicate 4 | 0.049 | 0.046 |
Fig.8 Raw Abs600 values(Colony 1)
We also used colony 2 series to do the same experiments. The features are similar to colony group. However, Abs600 value of device 2 reach the same level of device 3.
| Hour 0 | Hour 6 | ||
|---|---|---|---|
| Neg. Control | Replicate 1 | 0.071 | 0.285 |
| Replicate 2 | 0.068 | 0.285 | |
| Replicate 3 | 0.069 | 0.285 | Replicate 4 | 0.072 | 0.286 |
| Pos. Control | Replicate 1 | 0.068 | 0.299 | Replicate 2 | 0.067 | 0.296 | Replicate 3 | 0.071 | 0.297 | Replicate 4 | 0.071 | 0.303 |
| Device1 | Replicate 1 | 0.068 | 0.238 | Replicate 2 | 0.067 | 0.249 | Replicate 3 | 0.073 | 0.244 | Replicate 4 | 0.069 | 0.246 |
| Device2 | Replicate 1 | 0.065 | 0.295 | Replicate 2 | 0.064 | 0.299 | Replicate 3 | 0.069 | 0.301 | Replicate 4 | 0.07 | 0.306 |
| Device3 | Replicate 1 | 0.068 | 0.295 | Replicate 2 | 0.068 | 0.304 | Replicate 3 | 0.069 | 0.298 | Replicate 4 | 0.067 | 0.302 |
| Device4 | Replicate 1 | 0.069 | 0.217 | Replicate 2 | 0.075 | 0.22 | Replicate 3 | 0.068 | 0.218 | Replicate 4 | 0.067 | 0.217 |
| Device5 | Replicate 1 | 0.066 | 0.156 | Replicate 2 | 0.067 | 0.16 | Replicate 3 | 0.07 | 0.162 | Replicate 4 | 0.068 | 0.161 |
| Device6 | Replicate 1 | 0.064 | 0.268 | Replicate 2 | 0.067 | 0.281 | Replicate 3 | 0.065 | 0.269 | Replicate 4 | 0.069 | 0.272 |
| LB+Chior(blank) | Replicate 1 | 0.048 | 0.043 | Replicate 2 | 0.045 | 0.046 | Replicate 3 | 0.048 | 0.045 | Replicate 4 | 0.046 | 0.046 |
Fig.9 Raw Abs600 values(Colony 2)
2.2 Fluorescence
We used same colonies to measure their fluorescence values of device.
As it is shown in figure 8, Fluorescence value of device 1 is the highest while the value of device 3 is the lowest, which approaches to the value of counterpart of negative control.
| Hour 0 | Hour 6 | ||
|---|---|---|---|
| Neg. Control | Replicate 1 | 0.3566 | 0.3961 |
| Replicate 2 | 0.3743 | 0.4148 | |
| Replicate 3 | 0.3855 | 0.4076 | Replicate 4 | 0.3704 | 0.4111 |
| Pos. Control | Replicate 1 | 0.5663 | 1.337 | Replicate 2 | 0.5666 | 1.409 | Replicate 3 | 0.5281 | 1.396 | Replicate 4 | 0.5371 | 1.382 |
| Device1 | Replicate 1 | 0.7175 | 2.538 | Replicate 2 | 0.7509 | 2.789 | Replicate 3 | 0.7584 | 2.656 | Replicate 4 | 0.724 | 2.656 |
| Device2 | Replicate 1 | 0.6816 | 1.617 | Replicate 2 | 0.6791 | 1.735 | Replicate 3 | 0.6787 | 1.769 | Replicate 4 | 0.7568 | 1.776 |
| Device3 | Replicate 1 | 0.3702 | 0.4131 | Replicate 2 | 0.3774 | 0.4373 | Replicate 3 | 0.373 | 0.4332 | Replicate 4 | 0.391 | 0.4299 |
| Device4 | Replicate 1 | 0.5216 | 0.1.317 | Replicate 2 | 0.5383 | 1.387 | Replicate 3 | 0.5383 | 1.401 | Replicate 4 | 0.527 | 1.354 |
| Device5 | Replicate 1 | 0.4981 | 1.145 | Replicate 2 | 0.5158 | 1.228 | Replicate 3 | 0.5069 | 1.198 | Replicate 4 | 0.5063 | 1.194 |
| Device6 | Replicate 1 | 0.4791 | 0.8039 | Replicate 2 | 0.5 | 0.8388 | Replicate 3 | 0.482 | 0.8833 | Replicate 4 | 0.5077 | 0.7948 |
| LB+Chior(blank) | Replicate 1 | 0.3488 | 0.3416 | Replicate 2 | 0.3577 | 0.3606 | Replicate 3 | 0.3647 | 0.36 | Replicate 4 | 0.3229 | 0.3624 |
Fig.10 Raw Fluorescence values(Colony 1)
At the second time, results of almost devices are in agreement to the first time, nevertheless, fluorescence value for device 2, which going to the highest.
| Hour 0 | Hour 6 | ||
|---|---|---|---|
| Neg. Control | Replicate 1 | 0.3664 | 0.3724 |
| Replicate 2 | 0.3619 | 0.3871 | |
| Replicate 3 | 0.3478 | 0.4008 | Replicate 4 | 0.3743 | 0.4208 |
| Pos. Control | Replicate 1 | 0.5199 | 1.259 | Replicate 2 | 0.5257 | 1.31 | Replicate 3 | 0.5044 | 1.312 | Replicate 4 | 0.5163 | 1.323 |
| Device1 | Replicate 1 | 0.5493 | 1.791 | Replicate 2 | 0.5729 | 1.941 | Replicate 3 | 0.5377 | 1.89 | Replicate 4 | 0.5425 | 1.937 |
| Device2 | Replicate 1 | 0.629 | 2.095 | Replicate 2 | 0.615 | 2.142 | Replicate 3 | 0.5999 | 2.266 | Replicate 4 | 0.6153 | 2.303 |
| Device3 | Replicate 1 | 0.3725 | 0.4169 | Replicate 2 | 0.3825 | 0.4468 | Replicate 3 | 0.3815 | 0.4437 | Replicate 4 | 0.3767 | 0.4373 |
| Device4 | Replicate 1 | 0.5816 | 1.26 | Replicate 2 | 0.5933 | 1.299 | Replicate 3 | 0.5749 | 1.39 | Replicate 4 | 0.5997 | 1.344 |
| Device5 | Replicate 1 | 0.5378 | 1.042 | Replicate 2 | 0.5532 | 0.1.093 | Replicate 3 | 0.5525 | 1.081 | Replicate 4 | 0.5725 | 1.092 |
| Device6 | Replicate 1 | 0.4351 | 0.7296 | Replicate 2 | 0.4611 | 0.8149 | Replicate 3 | 0.4447 | 0.8102 | Replicate 4 | 0.4324 | 0.7783 |
| LB+Chior(blank) | Replicate 1 | 0.3476 | 0.3708 | Replicate 2 | 0.3606 | 0.3631 | Replicate 3 | 0.347 | 0.3567 | Replicate 4 | 0.3223 | 0.3567 |
Fig.11 Raw Fluorescence values(Colony 2)
Protocol:
We measured the Abs600 of our cell cultures and then used the appropriate ratio between Abs600 and OD600 to calculate the OD600 value. After getting the results, we diluted our overnight culture to OD600=1.0 for each culture and checked it. The data of Fluorescence per OD and Fluorescence per Particle at 0 and 6 hours is shown as below.
| OD600/Abs600 | 2.65 | |||||||
|---|---|---|---|---|---|---|---|---|
| uM Fluorescein/a.u. | 0.344 | |||||||
| uM Fluorescein/OD | ||||||||
| Hour 0: | Neg.Control | Pos.Control | Device1 | Device2 | Device3 | Device4 | Device5 | Device6 |
| Colony 1,Replicate 1 | 1.410 | 1.175 | 2.391 | 2.398 | 0.132 | 1.180 | 1.076 | 0.889 |
| Colony 1,Replicate 2 | 1.290 | 1.178 | 2.684 | 1.812 | 0.134 | 1.233 | 1.079 | 0.923 |
| Colony 1,Replicate 3 | 1.009 | 0.757 | 1.964 | 1.939 | 0.043 | 1.126 | 0.838 | 0.761 |
| Colony 1,Replicate 4 | 1.111 | 1.462 | 2.601 | 3.310 | 0.353 | 1.261 | 1.399 | 1.089 |
| Colony 2,Replicate 1 | 0.972 | 1.117 | 1.869 | 2.147 | 0.161 | 1.445 | 1.370 | 0.709 |
| Colony 2,Replicate 2 | 0.931 | 0.973 | 1.530 | 1.737 | 0.123 | 1.006 | 1.135 | 0.592 |
| Colony 2,Replicate 3 | 0.972 | 0.888 | 1.302 | 1.562 | 0.213 | 1.478 | 1.211 | 0.745 |
| Colony 2,Replicate 4 | 0.986 | 1.006 | 1.503 | 1.583 | 0.336 | 1.713 | 1.475 | 0.621 |
| Hour 6: | Neg.Control | Pos.Control | Device1 | Device2 | Device3 | Device4 | Device5 | Device6 |
| Colony 1,Replicate 1 | 0.028 | 0.512 | 1.461 | 0.738 | 0.024 | 0.762 | 0.891 | 0.240 |
| Colony 1,Replicate 2 | 0.029 | 0.562 | 1.810 | 0.796 | 0.032 | 0.778 | 1.032 | 0.261 |
| Colony 1,Replicate 3 | 0.025 | 0.565 | 1.682 | 0.823 | 0.032 | 0.790 | 0.953 | 0.283 |
| Colony 1,Replicate 4 | 0.025 | 0.553 | 1.671 | 0.808 | 0.029 | 0.735 | 0.914 | 0.235 |
| Colony 2,Replicate 1 | 0.001 | 0.450 | 0.945 | 0.887 | 0.024 | 0.663 | 0.770 | 0.207 |
| Colony 2,Replicate 2 | 0.013 | 0.491 | 1.008 | 0.912 | 0.042 | 0.698 | 0.830 | 0.249 |
| Colony 2,Replicate 3 | 0.024 | 0.492 | 0.999 | 0.967 | 0.045 | 0.775 | 0.803 | 0.263 |
| Colony 2,Replicate 4 | 0.035 | 0.488 | 1.025 | 0.971 | 0.041 | 0.749 | 0.829 | 0.242 |
Fig.12 Fluorescence per OD
| Particles/Abs600 | 3.29E+08 | |||||||
|---|---|---|---|---|---|---|---|---|
| MEFL/a.u. | 2.07E+12 | |||||||
| MEFL/particle | ||||||||
| Hour 0: | Neg.Control | Pos.Control | Device1 | Device2 | Device3 | Device4 | Device5 | Device6 |
| Colony 1,Replicate 1 | 6.84E+04 | 5.70E+04 | 1.16E+05 | 1.16E+05 | 6.41E+03 | 5.72E+04 | 5.22E+04 | 4.32E+04 |
| Colony 1,Replicate 2 | 6.26E+04 | 5.71E+04 | 1.30E+05 | 8.79E+04 | 6.52E+03 | 5.98E+04 | 5.24E+04 | 4.48E+04 |
| Colony 1,Replicate 3 | 4.90E+04 | 3.67E+04 | 9.53E+04 | 9.41E+04 | 2.09E+03 | 5.46E+04 | 4.07E+04 | 3.69E+04 |
| Colony 1,Replicate 4 | 5.39E+04 | 7.09E+04 | 1.26E+05 | 1.61E+05 | 1.71E+04 | 6.12E+04 | 6.79E+04 | 5.29E+04 |
| Colony 2,Replicate 1 | 4.71E+04 | 5.42E+04 | 9.07E+04 | 1.04E+05 | 7.83E+03 | 7.01E+04 | 6.65E+04 | 3.44E+04 |
| Colony 2,Replicate 2 | 4.52E+04 | 4.72E+04 | 7.42E+04 | 8.42E+04 | 5.99E+03 | 4.88E+04 | 5.51E+04 | 2.87E+04 |
| Colony 2,Replicate 3 | 4.72E+04 | 4.31E+04 | 6.32E+04 | 7.58E+04 | 1.03E+04 | 7.17E+04 | 5.88E+04 | 3.62E+04 |
| Colony 2,Replicate 4 | 4.69E+04 | 4.88E+04 | 7.29E+04 | 7.68E+04 | 1.63E+04 | 8.31E+04 | 7.16E+04 | 3.01E+04 |
| Hour 6: | Neg.Control | Pos.Control | Device1 | Device2 | Device3 | Device4 | Device5 | Device6 |
| Colony 1,Replicate 1 | 1.36E+03 | 2.49E+04 | 7.09E+04 | 3.58E+04 | 1.18E+03 | 3.70E+04 | 4.32E+04 | 1.16E+04 |
| Colony 1,Replicate 2 | 1.43E+03 | 2.73E+04 | 8.78E+04 | 3.86E+04 | 1.55E+03 | 3.78E+04 | 5.01E+04 | 1.26E+04 |
| Colony 1,Replicate 3 | 1.22E+03 | 2.74E+04 | 8.16E+04 | 3.99E+04 | 1.55E+03 | 3.83E+04 | 4.63E+04 | 1.37E+04 |
| Colony 1,Replicate 4 | 1.22E+03 | 2.68E+04 | 8.11E+04 | 3.92E+04 | 1.41E+03 | 3.57E+04 | 4.43E+04 | 1.14E+04 |
| Colony 2,Replicate 1 | 4.16E+01 | 2.18E+04 | 4.58E+04 | 4.31E+04 | 1.15E+03 | 3.22E+04 | 3.74E+04 | 1.00E+04 |
| Colony 2,Replicate 2 | 6.32E+02 | 2.38E+04 | 4.89E+04 | 4.42E+04 | 2.04E+03 | 3.38E+04 | 4.03E+04 | 1.21E+04 |
| Colony 2,Replicate 3 | 1.16E+03 | 2.39E+04 | 4.85E+04 | 4.69E+04 | 2.16E+03 | 3.76E+04 | 3.90E+04 | 1.27E+04 |
| Colony 2,Replicate 4 | 1.68E+03 | 2.37E+04 | 4.97E+04 | 4.71E+04 | 1.98E+03 | 3.63E+04 | 4.02E+04 | 1.17E+04 |
Fig.13 Fluorescence per Particle
Finally, we counted the colonies on each plate with fewer than 300 colonies. Then, we multiplied by the Final Dilution Factor: 8 . We recorded the CFU per 1mL of each OD600 = 0.1 culture.