Team:Amoy/Interlab

Aomy/Project

INTERLAB

1. Introduction

The goal of the Interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world. This year, three devices were cloned by ourselves, and one positive control and one negative control were provided by the registry. Using a plate reader, fluorescence measurements were obtained in arbitrary units. The results show increased fluorescence in the stronger promoter expected.

After the experiment, three required devices were created:
J23101+I13504 in the pSB3K3 backbone.
J23106+I13504 in the pSB1C3 backbone.
J23117+I13504 in the pSB1C3 backbone.

Devices constructed in pSB1C3 are high copy number plasmids, hence a strong fluorescence can be obviously observed by naked eyes. While for device J23117 + I13504, the promoter strength is the weakest, we could barely observe any fluorescence under natural light.

Figure 1. GFP generator with different promoters under natural light.
1. J23101+I13504 in DH5α;
2. J23106+I13504 in DH5α;
3. J23117+I13504 in DH5α.

Figure 2. Centrifugation of bacteria.
1. J23101+I13504;
2. J23106+I13504;
3. J23117+I13504;
4. Positive control (I20270);
5. Negative control (R0040 in pSB1C3)

Digestion Verification

Four devices (J23101+I13504/J23106+I13504/J23117+I13504/I20270) are double digested at EcoR I and Pst I restriction sites to verify the target parts. The restriction map is shown as Figure 3. As expected, the second bands are supposed to be aroud 1000bp, which is consistent with what we saw on the map.

Figure 3. Restriction map of digestion verification. 2000plus DNA marker.
1. Double digestion of J23101+I13504 (constructed plasmid)
2. Double digestion of J23106+I13504 (constructed plasmid)
3. Double digestion of J23117+I13504 (constructed plasmid)
4. Double digestion of I20270 in pSB1C3 backbone (provided in registry)

DNA sequencing

All created parts are verified by DNA sequencing, as shown in Figure 4.

Figure 4. DNA sequencing results of three required devices, analyzed by DNAMAN software.

2. Protocol

1. Transform constructed plasmids into DH5α competent cells, grown in incubator for 12 hrs at 37℃.
2. Add 5 mL LB medium with antibiotic (Chloramphenicol 35 μg/mL) into test tubes, choose monoclonal cells from the petri dish.
3. Set up 3 biological replicates of each device. Cultures were grown in test tubes for 16 hours at 37℃, shaking at 200 rpm.
4. Obtain initial OD 600 measurement of overnight cultures. Then dilute each sample to an OD of 0.5 (margin of error: 5%).
5. Set instrument to measure GFP.
6. Measurements of absorbance and fluorescence:
(1) OD 600
Device: Spectrophotometer
Wavelengths: 600 nm absorption.
(2) Fluorescence
Device: FLUOstar omega microplate reader, 96-well plates.
Wavelengths: 485 nm excitation, 520 nm emission.

3. Measurements

Absorbance at 600 nm was measured for each of the three cultures, alongside controls for media and contamination background. The dilution required for each sample has been calculated. Table 1 presents the re-measurement of samples on OD 600, and all OD 600 are within 5% of 0.5 (0.475-0.525).

Table 1. Absorbance at 600 nm for 3 required devices with different promoters as read in spectrophotometer.

A B C D E F
1 0.509 0.500 0.502 0.484 0.479 0.089
2 0.499 0.500 0.507 0.519 0.497 0.074
3 0.505 0.497 0.503 0.483 0.509 0.080
G / / / / / /

A wells=cloned device (J23101+I13504);
B wells=cloned device (J23106+I13504);
C wells=cloned device (J23117+I13504);
D wells=negative control (R0040 in pSB1C3 backbone);
E wells=positive control (I20270 in pSB1C3 backbone);
F wells=LB media plus antibiotic (chloramphenicol only);
G wells=Non-inoculate culture;

Table 2 presents the Fluorescence we measured, using three biological replicates of each device, alongside controls for LB media plus antibiotic and background value.

Table 2a. Fluorescence for 3 devices read in a FLUOstar omega microplate reader
J23101+I13504 J23106+I13504 J23117+I3504
A1 A2 A3 B1 B2 B3 C1 C2 C3
Replicate1 57850 55881 55501 36054 34549 34799 958 960 957
Replicate2 55682 54154 53233 37165 35969 35697 1098 1073 1071
Replicate3 56413 54693 53803 33475 31901 31920 1401 1381 1375
F 6937 6892 7183 / / / / / /
G 114 108 105 / / / / / /
Table 2b. Fluorescence of negative and positive control.
Negative Control Positive Control
D1 D2 D3 E1 E2 E3
Replicate1 802 785 777 10593 10104 10021
Replicate2 700 769 752 10758 10161 10136
Replicate3 1026 1022 1051 10990 10357 10357
F / / / / / /
G / / / / / /

A1-A3 wells=Cloned device J23101+I13504.
B1-B3 wells=Cloned device J23106+I13504.
C1-C3 wells=Cloned device J23117+I13504..
D1-D3 wells=Negative control device (only promoter).
E1-E3 wells=Positive control device (I20270 in PSB1C3 backbone);
F wells=LB media plus antibiotic (chloramphenicol only).
G wells=non-inoculate cultures
Three technical replicates (horizontal), three biological replicates(vertical) for A, B, C, and D.

Data was processed as follows:
1. Background absorbance was removed by subtracting the value of F wells.
2. Background fluorescence was controlled for by subtracting the value of F wells.
3. Calculate the mean value and standard deviation of the 3 biological replicates and 3 technical replicates.
4. Evaluate the variability of chosen analysis technique.

Table 3a. Fluorescence of 3 different constructs expressing GFP.
A1 A2 A3 B1 B2 B3 C1 C2 C3
Mean 56766 54909 54179 36610 34140 34139 1028 1138 1134
S. D. 1102.99 883.59 1179.83 1893.04 2064.66 1973.19 226.44 217.90 216.08
C. V. 1.94 1.61 2.18 5.17 6.05 5.78 22.02 19.15 19.05
Table 3b. Positive and negative controls of fluorescence
Negative Control Positive Control
D1 D2 D3 E1 E2 E3
Mean 751 859 860 10676 10207 10171
S. D. 166.76 141.68 165.88 199.44 132.71 170.76
C. V. 22.21 16.49 19.29 1.87 1.30 1.68
S. D.-standard deviation. C.V-coefficient of variation.

4. Provenance

Q: Who did the actual work to acquire these measurements?
A: Beibei Fang.

Q: What other people should be credited for these measurements?
A: Shouqiang Hong, Na Li.

Q: On what dates were the protocols run and the measurements taken?
A: Required devices were constructed by 30th June 2015. All samples were measured on 12th August 2015.

5. Appendix: Raw Data (Ex485 /Em520)

Technical replicate 1

1 2 3 4 5
A / / / mean /
B / / / / /
C 57850 55682 56413 56648 /
D 36054 37165 33475 35565 /
E 958 1098 1401 1152 /
F 802 700 1026 843 /
G 10593 10758 10990 10780 /
H 6937 6892 7183 7004 /

Technical replicate 2

1 2 3 4 5
A / / / mean /
B / / / / /
C 55881 54154 54693 55018 /
D 34549 35969 31901 35259 /
E 960 1073 1381 1017 /
F 785 769 1022 777 /
G 10104 10161 10357 10133 /
H 6802 6783 7072 6793 /

Technical replicate 3

1 2 3 4 5
A / / / mean stdv
B / / / / /
C 55501 53233 53803 54179 1180
D 34799 35697 31920 34139 1973
E 957 1071 1375 1134 1216
F 777 752 1051 860 166
G 10021 10136 10357 10171 171
H 6783 6762 6989 6845 125

6. Reference

1. Anderson,C., Berkley iGEM Team., (2006). Anderson Promoter Collection. Registry of Standard Biological Parts.
2. http://parts.igem.org/Promoters/Catalog/Anderson [Accessed 2014.09.19]
3. https://2014.igem.org/Team:XMU-China/Project_Interlab
4. https://2014.igem.org/Team:Imperial/InterLab_Study