Team:Hong Kong HKUST/InterLab
Team Hong Kong - HKUST
InterLab
What is Interlab?
InterLab Study is a measurement of GFP fluorescence among different lab organized by the iGEM community, aiming to produce common, comparable units for measuring GFP with different plate readers. This year (2017), 6 different RBS devices are tested.
Our Protocol
- Transformation: please refer here for the protocol.
- Calibration and Cell measurement: We were following the protocol provided except that we have measured the Abs595 instead of Abs600 due to the limitation of the plate reader in HKUST.
- Plate reader: Envision Xcite Multilabel Plate Reader
- Conversion from Abs595 to Abs600 (measured by spectrophotometer)
Fig. 1 Conversion of Abs595 to Abs600Parts and Materials:
- Machines:
EnVision multilabel reader (model: EnVision Xcite)
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Materials:
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 - Parts:
| Parts | Parts Location on Kit Plate | Parts used as the Promoter (strength) | Parts used as the RBS (efficiency) | Reporter Gene | Parts used as the Terminator | Backbone used |
|---|---|---|---|---|---|---|
| Positive Control (BBa_I20270) | Plate 6 Well 20B | BBa_J23151 (nil) | BBa_B0032 (0.3) | GFP | BBa_B0015 | pSB1C3 |
| Negative Control (BBa_R0040) | Plate 6 Well 20D | BBa_R0040 (nil) | nil | |||
| Test Device 1 (BBa_J364000) | Plate 6 Well 20F | BBa_J23101 (1791au) | BBa_B0034 (1.0) | |||
| Test Device 2 (BBa_J364001) | Plate 6 Well 20H | BBa_J23106 (1185au) | ||||
| Test Device 3 (BBa_J364002) | Plate 6 Well 20J | BBa_J23117 (162au) | ||||
| Test Device 4 (BBa_J364003) | Plate 6 Well 20L | BBa_J23101(1791au) | BBa_J364100* (nil) | |||
| Test Device 5 (BBa_J364004) | Plate 6 Well 20N | BBa_J23106(1185au) | ||||
| Test Device 6 (BBa_J364005) | Plate 6 Well 20P | BBa_J23117(162au) |
BBa_J364100 is a bicistronic design element (BCD2) that functions as a more controllable device to control translation comparing to prokaryotic RBS that may not be functionable with different coding sequences. In BCD, by ribosomes binding to the SD core sequence (SD1), the leader peptide will be synthesized, it gives SD1-directed ribosomes that recognizes and initiates translation via another SD site (SD2), following that, GOI (gene of interest) will be expressed.
Fig. 2 BCD diagram2Final Results
Fig. 3 uM Fluorescein/OD600 of Colony 1 of the 6 test device with a positive and a negative control.
Fig. 4 uM Fluorescein/OD600 of Colony 2 of the 6 test device with a positive and a negative control.
Fig. 5 uM Fluorescein/OD600 of the average of the 2 colonies of the 6 test device with a positive and a negative control.Conclusion
From the results obtained above, the promoters BBa_J23101, BBa_J23106 and BBa_J23117 has a decreasing promoter strength, while the RBS BBa_B0034 has a higher strength than BBa_J364100.
References
- The 2017 International Genetically Engineered Machine. (7 July, 2017). Tracks/Measurement/Interlab study/Plate Reader Protocol. Retrieved from https://2017.igem.org/Competition/InterLab_Study/Plate_Reader
- Mutalik, V. K., Guimaraes, J. C., Cambray, G., Mai, Q. A., Christoffersen, M. J., Martin, L., et al. (2013). Precise and reliable gene expression via standard transcription and translation initiation elements. Nature Methods, 10, 354–360.
