Aomy/Project

Interlab

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 35μg/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 coding for GFP as read in spectrophotometer. Units are arbitrary.
	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

Table1:Absorbance at 600 nm for 3 required devices with different promoters coding for GFP as read in spectrophotometer. Units are arbitrary.

'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.

	A1	A2	A3	B1	B2	B3	C1	C2	C3	D1	D2	D3	E1	E2	E3
replicate1	57850	55881	55501	36054	34549	34799	958	960	957	802	785	777	10593	10104	10021
replicate2	55682	54154	53233	37165	35969	35697	1098	1073	1071	700	769	752	10758	10161	10136
replicate3	56413	54693	53803	33475	31901	31920	1401	1381	1375	1026	1022	1051	10990	10357	10357
F	6937	6892	7183
G	114	108	105

Table2:Fluorescence(485 ex/520 em)for 3 devices with different promoters coding for GFP as read in a FLUOstar omega microplate reader. Units are arbitrary.

'A1-A3'wells=Cloned device J23101+I13504. Three technical replicates (horizontal), three biological replicates(vertical).
'B1-B3'wells=Cloned device J23106+I13504. Three technical replicates (horizontal), three biological replicates (vertical).
'C1-C3'wells=Cloned device J23117+I13504. Three technical replicates (horizontal), three biological replicates (vertical).
'D1-D3'wells=negative control device (only promoter). Three technical replicates (horizontal), three biological replicates (vertical).
'E1-E3'wells=positive control device (I20270 in PSB1C3 backbone);
'F'wells=LB media plus antibiotic (chloramphenicol only). Sampled three times.
'G'wells=non-inoculate cultures

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.

	A1	A2	A3	B1	B2	B3	C1	C2	C3	D1	D2	D3	E1	E2	E3
mean	56766	54909	54179	36610	34140	34139	1028	1138	1134	751	859	860	10676	10207	10171
S.D.	1102.99	883.59	1179.83	1893.04	2064.66	1973.19	226.44	217.90	216.08	166.76	141.68	165.88	199.44	132.71	170.76
C.V.	1.94	1.61	2.18	5.17	6.05	5.78	22.02	19.15	19.05	22.21	16.49	19.29	1.87	1.30	1.68

Table 3. Comparison of fluorescence in terms of three technical replicates for three different constructs expressing GFP. D1-D3-negtive controls, E1-E3-positive controls.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 30^th June 2015. All samples were measured on 12^th August 2015.

5. Appendix

Raw Data (Ex485/Em520)

Technical replicate1:

	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 replicate2:

	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 replicate3:

	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 19/09/2014]
3. https://2014.igem.org/Team:XMU-China/Project_Interlab
4. https://2014.igem.org/Team:Imperial/InterLab_Study

Team:Amoy/Interlab