Interlab Study

Motivation

Fluorescence measurements usually cannot be compared as they are reported in different units or being processed in different ways. Often we work around this by doing “relative expression” comparison. However, a lack of standard makes it harder to debug engineered biological constructs, harder to effectively share constructs between labs, and harder even to just interpret your experimental controls.

Experimental Design

Interlab study for 2016 iGEM aimed to quantify expression of five different reporter constructs which express GFP under the control of different promoter and RBS parts. The study focuses on the standardization of fluorescence measurements and primarily involves measurement of fluorescencea and OD600 values. It has been divided into 4 modules: OD600 reference point, FITC standard curve, Normalization, Cell measurement.

We received interlab kit, which had 7 tubes (5 with plasmid DNA and 3 with standard reagents):

1. Plasmid DNA (100 pg/uL in 10uL of Buffer EB)

2. Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3

3. Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3

4. Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3

5. Positive Control Device: I20270 in pSB1C3

6. Negative Control Device: R0040 in pSB1C3

FITC Standard: one tube with dried down FITC for creating a FITC standard

LUDOX: one tube with 30% colloidal silica suspended in 1mL of water

We stored these tubes at 4C.

Instrumentation

We used Tecan Infinite M200 Pro instrument to measure OD600 and fluorescent values. GFP and FITC sample readings were taken in NUN96ft Black Thermo plates. The instrument settings were kept as: Excitation/Emission wavelengths-481nm/511nm, Bandwidth-9/20, Gain-Manual(100), Flashes-25, Integration time-20. Sample volume was 100ul. OD600 sample measurements were taken in Tarsons 96 well micro test plate (Cat. No. 941196). Sample volume was 100ul.

Protocol

Standard iGEM 2016 Interlab study protocol was used.

Results

Our results for interlab study can be found here.

Conclusion

We performed all the experiments as it was mentioned in the guideline, given by iGEM 2016 HQ. To our and iGEM hq's surprise, we found that E. coli DH5alpha cells with test device 1 were growing very slow in comparison to other test devices in secondary culture. At the end of 16th hour of our primary culture, we found the OD values for each test device to be similar. Although, cells with test device 1 were growing very slow, they showed gfp expressions.

Attributions

Nitish worked on transformation and sample collection. Venkat took measurements and analysed the results.