Team:Toronto/InterLab
Interlab
Description
iGEM Toronto was excited to participate in the 2017 Interlab Study to help establish a GFP fluorescence measurement protocol and characterize important promoters and RBS devices. In science and engineering, standardization is an important key to generating reliable data and comparing results from different labs. By comparing our data with labs from around the world, iGEM will be able to establish common standards of measurement for GFP fluoresecence with different plate readers.
Plasmids
iGEM HQ provided 8 Interlab parts in total, all in the pSB1C3 backbone. They included a positive control, a negative control, and 6 test devices which contained various combinations of three promoters and two RBS devices. The positive control contained BBa_J23151, a 35 bp constitutive promoter, and BBa_B0032, a 13 bp weak RBS, while the negative control consisted only of BBa_R0040, a 54 bp TetR repressible promoter.
All the parts:
- BBa_E0040: a 720 bp GFP gene from the Aequeora victoria jellyfish
- Fluorescence is expected within 8 minutes
- Excitation max: 501 nm
- Emission max: 511 nm
- BBa_B0010: an 80 bp transcriptional terminator from E. coli rrnB with 64 bp stem loop
- BBa_B0012: a 41 bp transcriptional terminator from coliphageT7 for E. coli RNA Polymerase
- Not a good terminator
- promoter in reverse direction
The test devices were all 918 bp plasmids which constitutively express GFP. All consist of:
- A 35 bp constitutive promoter in the BBa_J23100-J23119 family
- 1 and 4 have BBa_J23101
- 2 and 5 have BBa_J23106 - medium strength; more characterization info from Sheffield 2016
- 3 and 6 have BBa_J23117
- A 12 bp RBS,
- 1-3 have BBa_B0034, a 12 bp RBS
- 4-6 have BBa_J364100, an 84 bp bicistronic design element (BCD2)
- BBa_E0040
- BBa_B0010
- BBa_B0012
| Part | Part number | Length (bp) | Promoter | RBS | Reporter Protein | Terminators |
|---|---|---|---|---|---|---|
| Positive control | BBa_I20270 | 919 | BBa_J23151 | BBa_B0032 | BBa_E0040 (GFP) | BBa_B0010 and BBa_B0012 |
| Negative control | BBa_R0040 | 54 | N/A | N/A | N/A | N/A |
| Test device 1 | BBa_J364000 | 918 | BBa_J23101 | BBa_B0034 | BBa_E0040 (GFP) | BBa_B0010 and BBa_B0012 |
| Test device 2 | BBa_J364001 | 918 | BBa_J23106 | BBa_B0034 | BBa_E0040 (GFP) | BBa_B0010 and BBa_B0012 |
| Test device 3 | BBa_J364002 | 918 | BBa_J23117 | BBa_B0034 | BBa_E0040 (GFP) | BBa_B0010 and BBa_B0012 |
| Test device 4 | BBa_J364003 | 918 | BBa_J23101 | BBa_J364100 | BBa_E0040 (GFP) | BBa_B0010 and BBa_B0012 |
| Test device 5 | BBa_J364004 | 918 | BBa_J23106 | BBa_J364100 | BBa_E0040 (GFP) | BBa_B0010 and BBa_B0012 |
| Test device 6 | BBa_J364005 | 918 | BBa_J23117 | BBa_J364100 | BBa_E0040 (GFP) | BBa_B0010 and BBa_B0012 |
