Part:BBa_K1412716
GFP generator with J23101
This part consists of Anderson promoter J23101 and GFP generator BBa_E0240. It can be used for characterization of promoter J23101. When in backbone vector pSB3K3, the copies is low, so we need observe it carefully. While in backbone vector pSB1C3, the expression is strong, so we can observe it easily.
Why we build this BioBrick?
When we want to measure the expression intensity of a Promoter, the most concise method is connect it with a GFP generator. Then we just need an device to measure the fluorescent, and compare their fluorescent intensity with each other.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 706
Protocol
1. Transformed BBa_k1412716 into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37℃.
2. Inoculate two 5 ml cultures of supplemented LB medium and antibiotic (Kanamycin 50 μg/ml) with single colony from the plate.
3. Cultures were grown in conical flask for 16 hrs at 37℃ with shaking at 200 rpm in the table concentrator.
4. Cultures were diluted 1:100 into 20 ml fresh LB medium and grown for 3 hrs at 37℃ with shaking at 200 rpm in the table concentrator.
5. Then the culture was spun down and washed twice with phosphate-buffered saline (PBS, pH 7.4) to minimize the background fluorescence from the medium.
6. The washed cells were suspended in PBS and diluted to bring the cells into an appropriate concentration range (2–5 times) before taking fluorimeter measurements.
7. Measure the fluorescence and absorbance:
(1)Fluorescence:
Device: SpectraMax+M5 microplate reader, 96-well plates.
Wavelengths: 501 nm excitation, 514 nm emission, Auto-cutoff: 515 nm.
(2)OD600 (optical density at 600 nm):
Device: SpectraMax+M5 microplate reader, 96-well plates.
Wavelengths: 600 nm absorption.
8. Measure every 30 minutes in the next 4 hrs.
References
[1] Bagh, Sangram, Mahuya Mandal, and David R. McMillen. "Minimal genetic device with multiple tunable functions." Physical Review E 82.2 (2010): 021911. Available from: http://journals.aps.org/pre/abstract/10.1103/PhysRevE.82.021911.
More information, click here: http://2014.igem.org/Team:XMU-China#
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