INTRODUCTION

InterLab Measurement Study, an important element under the Bronze Medal criteria of the iGEM competition strives to standardize measurements and data processing across various lab environments thus addressing one of the major hurdles in the field of synthetic biology and engineering. This enables scientists to calibrate the defect in engineered biological construct, to share construct between labs and to analyse experimental results across different lab conditions. The iGEM Measurement Committee, through the InterLab study, has been developing a robust and resolute measurement procedure for the Green Fluorescent Protein (GFP) over the span of three years. GFP is widely used owing to its ability to report gene expression and successful transformation. Our role in this study required that we submit measurement data dealing with the fluorescence of GFP and Optical Density(OD) associated with cells transformed with different test devices.

Click here to download the interlab study protocol

TEST DEVICES

6 Test Devices, a Positive Control and a Negative Control were provided, all of which contained the Chloramphenicol resistance genebuilt in a pSB1C3 vector. Additionally all devices except the negative control had built-in GFP. The negative control had Tet R which is non-fluorescent.

The devices contained 3 different promoters of the Anderson family. Those were,
1. Bba_J23101 which has high expression
2. Bba_J23106 which has medium expression
3. Bba_J23117 which has low expression.

Test Device 1:

pSB1C3 backbone containing J23101 (Promoter), B0034 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)

Test Device 2:

pSB1C3 backbone containing J23106 (Promoter), B0034 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)

Test Device 3:

pSB1C3 backbone containing J23117 (Promoter), B0034 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)

Test Device 4:

pSB1C3 backbone containing J23101 (Promoter), J364100 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)

Test Device 5:

pSB1C3 backbone containing J23106 (Promoter), J364100 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)

Test Device 1:

pSB1C3 backbone containing J23117 (Promoter), J364100 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)

Positive control

pSB1C3 backbone containing J23151 (Promoter), B0032 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)

Negative control

pSB1C3 backbone containing R0040(no GFP expression device)

RESULTS

OD600 CALIBRATION GRAPH

Replicate 1	0.053	0.047
Replicate 2	0.064	0.041
Replicate 3	0.063	0.041
Replicate 4	0.052	0.036
Arith. Mean	0.058	0.04125
Corrected Abs600	0.01675
Reference OD600	0.0425
OD600/Abs600	2.537313433

FLUORESCEIN STANDARD CURVE

uM Fluorescein	50.00	25	12.5	6.25	3.125	1.5625	0.78125	0.390625	0.195313	0.097656	0.048828	0
Replicate 1	519204	519217	520637	520277	520367	430585	245987	184837	111646	58046	30246	321
Replicate 2	529594	529958	530951	529430	530035	529679	373525	243644	123362	72848	37522	244
Replicate 3	530928	530493	531124	530361	530215	518343	349581	223415	132286	83477	36720	291
Replicate 4	534732	534876	534415	535752	534146	518933	362986	234241	137938	78760	39434	328
Arith. Mean	528614.5	528636	529281.8	528955	528690.8	499385	333019.8	221534.3	126308	73282.75	35980.5	296
Arith. Std.Dev.	6640.608	6654.605	5979.472	6421.561	5864.438	46161.66	58843.37	25823.3	11469.47	11049.44	3988.911	38.20122

OD Readings

RAW PLATE READER MEASUREMENT

FLUORESCENCE MEASUREMENT

FLUORESCENCE READING

CONCLUSION

The Plate Reader results indicate that Test Device 4 showed highest GFP expression and thus it has the strongest promoter and the best RBS. This is in accordance with Anderson promoter collections which establishes that Bba_J23101 has the highest GFP expression of the three promoters provided.The other devices produced results (absence of significant GFP expression) which indicates weak or non-functional promoters and RBS.

Team:REC-CHENNAI/InterLab

INTRODUCTION

TEST DEVICES

Test Device 1:

Test Device 2:

Test Device 3:

Test Device 4:

Test Device 5:

Test Device 1:

Positive control

Negative control

RESULTS

OD600 CALIBRATION GRAPH

FLUORESCEIN STANDARD CURVE

OD Readings

RAW PLATE READER MEASUREMENT

FLUORESCENCE MEASUREMENT

FLUORESCENCE READING

CONCLUSION