Team:Hong Kong HKUST/Interlab
Interlab
We participated in the 2016 iGEM InterLab Measurement Study which aims to improve the common tools that are used by both the iGEM and synthetic biology communities. A ‘relative expression’ comparison on green fluorescence measurement with a particular unit and protocol was performed.
PROTOCOL
The protocol for plate reader stated in the iGEM Headquarter was used. All details were followed except for the OD measurement and the E. coli strain in use. OD595 was employed rather than OD600 due to the limitation of the plate reader in the HKUST. E. coli strain DH10B which is highly similar to Top 10 was used.
Calibrations of the machine, EnVision multilabel reader, and the OD595 reference point were done before the measurement.
Parts and Materials
An InterLab kit was provided by the iGEM headquarter.
| Parts | Materials | Machines |
|---|---|---|
| Test device 1: pSB1C3-BBa_J23101-BBa_I13504 |
FITC Standard: one tube with dried down FITC for creating a FITC standard |
EnVision multilabel reader (model: EnVision Xcite) |
| Test device 2: pSB1C3-BBa_J23106-BBa_I13504 |
LUDOX: one tube with 30% colloidal silica suspended in 1mL of water |
Filter used for measuring GFP: FITC |
| Test device 3: pSB1C3-BBa_J23117-BBa_I13504 |
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| Positive control device: pSB1C3-BBa_I20271 |
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| Negative control device: pSB1C3-BBa_R0040i |
*To know more about the setting of EnVision multilabel reader, please click.
RESULTS
*To know more about the full version of measurement results, please download.
CONCLUSIONS
From the results obtained through the measurement, device 1 showed the highest overall green fluorescence level while device 3 showed the lowest fluorescence. This outcome was mainly due to the differences of the promoters of the three constructs. With the strongest promoter, BBa_J23101, the GFP produced by device 1 would be the highest among all three. Though all the three promoters are from the same constitutive promoter family, their promoter strengths are different. According to the Part Registry, the promoter strengths of BBa_J23101, BBa_J23106 and BBa_J23117 are 1791 au, 1185 au and 162 au respectively. This could be the main reason for causing the great difference of the GFP produced. Overall, the results obtained was reasonable.
REFERENCE
- The 2016 International Genetically Engineered Machine. (27 June, 2016). Tracks/Measurement/Interlab study/Plate Reader Protocol. Retrieved August 15, 2016 from https://2016.igem.org/Tracks/Measurement/Interlab_study/Plate_Reader_Protocol
- Registry of Standard Biological Parts. (4 August, 2006). Part: BBa_J23101. Retrieved, 2016 from http://parts.igem.org/Part:BBa_J23101
