Team:Fudan/InterLab

2018 iGEM Team:Fudan - InterLab

iGEM interLab

This year, we had the pleasure of taking part in iGEM’s Fifth International InterLaboratory Measurement Study in synthetic biology.

iGEM interLab

This year, we had the pleasure of taking part in iGEM’s Fifth International InterLaboratory Measurement Study in synthetic biology.

This year, we had the pleasure of participating in iGEM’s Fifth International InterLaboratory Measurement Study in synthetic biology. As taking reliable and repeatable measurements is crucial in synthetic biology, the Measurement Committee has been using the InterLab Study to develop a powerful and accurate measurement procedure for green fluorescent protein (GFP) by measuring it in absolute fluorescence units calibrated against a known concentration of fluorescent molecules. Nevertheless, a new problem emerges when we take bulk measurements of cell populations as the number of cells in the sample remains a large source of variability. Thus, the aim of this year’s Interlab study is to determine the cell count in each sample to remove the variability of cell populations in measurements of different labs. More concretely, we hope to discover if this can be achieved by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD.

Regarding experimental procedures, we used two orthogonal approaches to calculate the cell count in our samples:
1. Using silica beads to convert absorbance of cells into absorbance of a known concentration of beads.
2. Counting colony-forming units (CFUs) from the sample

We are first required to make three sets of unit calibration measurements: an OD600 reference point, particle standard curve, and a fluorescein standard curve. It is also important that we use the same plates, volumes, and settings as what we will use for our cell-based assays for the calibration measurements. The plate reader we used was the Biotek Cytation 3.

Calibration 1:​
OD​600​ Reference point - LUDOX Protocol

This calibration is to allow us to obtain a conversion factor which allows us to transform our absorbance (Abs600) data from the plate reader into a comparable OD600 measurement. This conversion is necessary as measurements of absorbance are dependent on volume, and the path length of the light defined by the fluid in the wells of the plate reader is unfixed and can vary from well to well.

Below is the data we obtained:

LUDOX CL-X H2O
Replicate 1 0.052 0.037
Replicate 2 0.051 0.038
Replicate 3 0.05 0.037
Replicate 4 0.052 0.037
Arith. Mean 0.051 0.037
Corrected Abs600 0.014
Reference OD600 0.063
OD600/Abs600 4.5
Table 1. From the measured replicates, the conversion factor of 4.5 is recorded for our 96-well plate reader for the 100 μL of LUDOX CL-X to water.

Calibration 2:​
Particle Standard Curve - Microsphere Protocol

For the second calibration, we performed a series of dilutions for the monodisperse silica microsphere and measured their Abs600 in the plate reader. A standard curve of particle concentration was also constructed to convert Abs600 measurements to a cell number estimate.

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 0.631 0.387 0.199 0.088 0.065 0.051 0.046 0.043 0.04 0.04 0.039 0.037
Replicate 2 0.73 0.389 0.2 0.105 0.074 0.07 0.048 0.042 0.045 0.038 0.039 0.039
Replicate 3 0.709 0.343 0.185 0.12 0.065 0.055 0.052 0.046 0.04 0.04 0.038 0.039
Replicate 4 0.896 0.338 0.221 0.116 0.074 0.056 0.049 0.043 0.042 0.04 0.039 0.038
Arith. Mean 0.742 0.364 0.201 0.107 0.07 0.058 0.049 0.044 0.042 0.04 0.039 0.038
Arith. Std.Dev. 0.111 0.028 0.015 0.014 0.005 0.008 0.003 0.002 0.002 0.001 0.001 0.001
Arith. Net Mean 0.703 0.326 0.163 0.069 0.031 0.02 0.011 0.005 0.004 0.001 0.001
Mean particles / Abs600 3.35e+08 3.61e+08 3.61e+08 4.26e+08 4.71e+08 3.72e+08 3.5e+08 3.5e+08 2.63e+08 3.68e+08 4.6e+08
Table 2. Measurement of Abs600 of the serial dilution of monodisperse silica microspheres
Figure1. A standard curve of Particle Count (100 μL) vs Abs600 graph
Figure 2. The log scale of a standard curve of Particle Count (100 μL) vs Abs600 graph

The log scale of the standard curve alters the originally relatively constant slanted line into a more exponential curve.

Calibration 3:​
Fluorescence standard curve - Fluorescein Protocol

For the third calibration, we hope to create a standard fluorescence curve in order to enable different teams to compare their fluorescence outputs. Therefore, we will prepare a serial dilution of fluorescein in four replicates and measure its fluorescence in a 96 well plate in the plate reader. Once measured, we can construct a standard fluorescence curve and use it to convert our cell-based readings to a corresponding fluorescein concentration.

Fluorescein μM 10 5 2.5 1.25 0.625 0.313 0.156 0.078 0.039 0.0195 0.0098 0
Replicate 1 50884 26269 13818 6944 3590 1802 891 459 219 120 61 1
Replicate 2 46604 26430 12604 7114 3614 1812 911 465 225 113 56 2
Replicate 3 53518 26706 13814 6902 3550 1778 887 443 229 122 61 1
Replicate 4 53094 26915 13384 7225 3650 1611 962 513 236 127 71 2
Arith. Mean 51000 26600 13400 7050 3600 1750 913 470 227 121 62.3 1.5
Arith. Std.Dev. 3170 287 572 150 42 94.3 34.5 30.1 7.14 5.8 6.29 0.577
Arith. Net Mean 51000 26600 13400 7050 3600 1750 911 469 226 119 60.8
μM Fluorescein/a.u. 0.000196 0.000188 0.000187 0.000177 0.000174 0.000179 0.000171 0.000167 0.000173 0.000164 0.000161
Mean μM fluorescein / a.u.: 0.000196
MEFL / a.u.: 0.000196
Table 4. Fluorescein (uM) measurement of serial dilution of fluorescein in four replicates.
Figure 3. Fluorescein Standard Curve of Fluorescein Concentration (uM) to Fluorescence
Figure 4. Fluorescein Standard Curve (log scale) of Fluorescein Concentration (uM) to Fluorescence

For the fluorescence standard curve, we used a gain setting of 50 and a filter that passes a light wavelength of 528 nm / 20 nm. We had an emission wavelength of 525 and an excitation wavelength of 488.

Cell Measurement

We used E. coli K-12 DH5-alpha for our cell measurements and maintained all the same plates, volumes, and settings in our calibration process to ensure that the measurements are valid.

Hour 0: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 117 209 149 228 125 256 139 136 127
Colony 1, Replicate 2 111 186 140 255 122 256 136 133 129
Colony 1, Replicate 3 109 209 134 248 114 212 139 147 113
Colony 1, Replicate 4 115 215 142 258 115 218 127 141 119
Colony 2, Replicate 1 113 227 136 267 110 258 148 131 133
Colony 2, Replicate 2 119 222 118 260 99 264 160 140 113
Colony 2, Replicate 3 115 237 129 262 111 246 149 138 114
Colony 2, Replicate 4 115 201 139 260 115 245 152 133 116
Table 6. Fluorescence Raw Readings at 0 hour
Hour 6: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 136 570 943 928 148 407 290 304 129
Colony 1, Replicate 2 134 518 920 897 150 421 266 303 141
Colony 1, Replicate 3 141 540 916 844 148 377 256 285 127
Colony 1, Replicate 4 129 544 927 850 140 389 277 278 136
Colony 2, Replicate 1 116 584 970 804 132 325 728 284 129
Colony 2, Replicate 2 134 567 889 726 131 332 710 298 132
Colony 2, Replicate 3 123 593 970 820 131 326 616 281 120
Colony 2, Replicate 4 129 604 945 846 131 327 759 303 124
Table 7. Fluorescence Raw Readings at 6 hours
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 0.056 0.047 0.047 0.053 0.054 0.05 0.045 0.052 0.039
Colony 1, Replicate 2 0.053 0.047 0.047 0.055 0.059 0.048 0.046 0.051 0.04
Colony 1, Replicate 3 0.054 0.05 0.047 0.053 0.058 0.05 0.048 0.054 0.039
Colony 1, Replicate 4 0.059 0.049 0.05 0.055 0.064 0.047 0.048 0.052 0.039
Colony 2, Replicate 1 0.056 0.053 0.05 0.058 0.062 0.054 0.056 0.057 0.041
Colony 2, Replicate 2 0.061 0.052 0.045 0.058 0.059 0.049 0.05 0.055 0.091
Colony 2, Replicate 3 0.057 0.051 0.045 0.057 0.057 0.048 0.051 0.054 0.04
Colony 2, Replicate 4 0.055 0.061 0.046 0.057 0.059 0.045 0.045 0.05 0.041
Table 8. Abs600 Raw Readings at 0 hour
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 0.356 0.211 0.296 0.344 0.359 0.052 0.403 0.282 0.039
Colony 1, Replicate 2 0.371 0.195 0.311 0.351 0.368 0.051 0.41 0.274 0.041
Colony 1, Replicate 3 0.377 0.211 0.319 0.35 0.362 0.05 0.412 0.282 0.042
Colony 1, Replicate 4 0.36 0.209 0.321 0.342 0.357 0.054 0.41 0.277 0.045
Colony 2, Replicate 1 0.35 0.193 0.31 0.336 0.356 0.051 0.389 0.294 0.04
Colony 2, Replicate 2 0.37 0.21 0.288 0.308 0.366 0.05 0.384 0.278 0.04
Colony 2, Replicate 3 0.356 0.218 0.315 0.342 0.361 0.051 0.346 0.296 0.039
Colony 2, Replicate 4 0.359 0.215 0.303 0.338 0.338 0.05 0.373 0.307 0.04
Table 9. Abs600 Raw Readings at 6 hours
Unit Scaling Factor
OD600 / Abs600 4.5
μM Fluorescein / a.u. 1.81E-04
Particles / Abs600 3.98E+08
MEFL / a.u. 1.09E+09
Table 10.

Regarding the observations during the experiment, it should be noted that our LB medium had an innately low fluorescence intensity. Also, from the data, we observed that the Abs600 growth for Device 4 was limited but it is still possible to test its fluorescence. Moreover, regarding the dilution of target Abs600 of 0.02, we first measured the 1:8 dilution of the overnight cultures and using the equations provided in the protocol, we calculated the amounts of source and LB needed to obtain the target Abs600 of 0.02.

Protocol:
Colony Forming Units per 0.1 OD600 E. coli cultures

For the CFU protocol, which is used to calibrate OD600 to colony forming unit counts, we have 2 Positive Control cultures and 2 Negative Control cultures. Our goal is to get the colony forming units (CFU) per 1mL of an OD600 = 0.1 culture.

OD600 OD600
Positive Control 0.247 0.254
Negative Control 0.31 0.326
Blank media 0.042 0.042
Table 11. OD600 values of Positive Control and Negative Control with Blank media.

Using the equation provided in the protocol, we calculated how much culture and media we should add to dilute the OD600 of our overnight culture to 0.1 in 1mL of LB + Cam media.

Culture amount needed (μL) Media amount needed (μL)
Positive Control Culture 1 487.8049 512.1951
Positive Control Culture 2 471.6981 528.3019
Negative Control Culture 1 373.1343 626.8657
Negative Control Culture 2 352.1127 647.8873
Table 12.
Control Culture 1 Control Culture 2 Starting Sample Dilutions for Culture 1.1 Starting Sample Dilutions for Culture 1.2 Starting Sample Dilutions for Culture 1.3 Starting Sample Dilutions for Culture 2.1 Starting Sample Dilutions for Culture 2.2 Starting Sample Dilutions for Culture 2.3
Positive Control Culture 0.272 0.279 0.12 0.121 0.119 0.12 0.116 0.123
Negative Control Culture 0.337 0.336 0.128 0.133 0.135 0.122 0.129 0.127
Blank media 0.042 0.043
Table 13. OD600 values of starting Sample Dilutions for Positive and Negative Control Cultures to ensure that OD600 is near 0.1
Dilution 4 (# colonies) Dilution 4 (CFU per 1mL of an OD600 =0.1 culture )
BBa_I20270 Culture 1, Dilution Replicate 1 19 1.52 x 10^7 CFU/mL
BBa_I20270 Culture 1, Dilution Replicate 2 25 2 x 10^7 CFU/mL
BBa_I20270 Culture 1, Dilution Replicate 3 30 2.4 x 10^7 CFU/mL
BBa_I20270 Culture 2, Dilution Replicate 1 42 3.36 x 10^7 CFU/mL
BBa_I20270 Culture 2, Dilution Replicate 2 53 4.24 x 10^7 CFU/mL
BBa_I20270 Culture 2, Dilution Replicate 3 54 4.32 x 10^7 CFU/mL
BBa_R0040 Culture 1, Dilution Replicate 1 130 1.04 x 10^8 CFU/mL
BBa_R0040 Culture 1, Dilution Replicate 2 106 8.48 x 10^7 CFU/mL
BBa_R0040 Culture 1, Dilution Replicate 3 95 7.6 x 10^7 CFU/mL
BBa_R0040 Culture 2, Dilution Replicate 1 89 7.12 x 10^7 CFU/mL
BBa_R0040 Culture 2, Dilution Replicate 2 102 8.16 x 10^7 CFU/mL
BBa_R0040 Culture 2, Dilution Replicate 3 104 8.32 x 10^7 CFU/mL
Table 14. Number of colonies of BBa_R0040 Culture and BBa_I20270 Culture of Dilution 4 and its corresponding CFU per 1mL of an OD600 = 0.1 culture

We successfully calibrated our OD600 to colony forming unit counts as we were fairly accurate in ensuring that the OD600 values were close to 0.1, which we believed was the most crucial part of the CFU protocol. Moreover, we utilized the data from dilution 4 for the conversion because it has the best countable number which is between 50 to 200 colonies.

We were fairly successful in completing the Interlab project as it was accepted by the iGEM committee the first time we submitted it. It was really quite an enjoyable side project to perform and to practice our experimental techniques in the lab.

2018 team Fudan abstract

Abstract

Contact-dependent signaling is critical for multicellular biological events, yet customizing contact-dependent signal transduction between cells remains challenging. Here we have developed the ENABLE toolbox, a complete set of transmembrane binary logic gates. Each gate consists of 3 layers: Receptor, Amplifier, and Combiner. We first optimized synthetic Notch receptors to enable cells to respond to different signals across the membrane reliably. These signals, individually amplified intracellularly by transcription, are further combined for computing. Our engineered zinc finger-based transcription factors perform binary computation and output designed products. In summary, we have combined spatially different signals in mammalian cells, and revealed new potentials for biological oscillators, tissue engineering, cancer treatments, bio-computing, etc. ENABLE is a toolbox for constructing contact-dependent signaling networks in mammals. The 3-layer design principle underlying ENABLE empowers any future development of transmembrane logic circuits, thus contributes a foundational advance to Synthetic Biology.