Difference between revisions of "Part:BBa K1412716"
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<partinfo>BBa_K1412716 short</partinfo> | <partinfo>BBa_K1412716 short</partinfo> | ||
| − | This part consists of Anderson promoter J23101 which is from the Anderson collection without RFP in backbone vector | + | This part consists of Anderson promoter J23101 which is from the Anderson collection without RFP in backbone vector pSB13K3 to easily fuse the promoter with other reporters e.g. the lux operon or the lacZ reporter gene. This part was also evaluated in the publication The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis by Radeck et al.. |
| + | == '''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. | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
<!-- --> | <!-- --> | ||
| − | <span class='h3bb'>Sequence and Features</span> | + | <span class='h3bb'> |
| − | <partinfo> | + | =='''Sequence and Features'''== |
| + | </span> | ||
| + | <partinfo>BBa_K1412924 SequenceAndFeatures</partinfo> | ||
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===Protocol=== | ===Protocol=== | ||
| − | 1. Streak a plate of the strain which contain one of the parts listed in | + | 1. Streak a plate of the strain which contain one of the parts listed in pSB3K3 . |
2. Inoculate two 5ml cultures of supplemented LB Medium and antibiotic (Chloromycetin 0.1mg/ml) with single colony from the plate. | 2. Inoculate two 5ml cultures of supplemented LB Medium and antibiotic (Chloromycetin 0.1mg/ml) with single colony from the plate. | ||
Revision as of 13:32, 2 September 2014
GFP generator with J23101
This part consists of Anderson promoter J23101 which is from the Anderson collection without RFP in backbone vector pSB13K3 to easily fuse the promoter with other reporters e.g. the lux operon or the lacZ reporter gene. This part was also evaluated in the publication The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis by Radeck et al..
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. Streak a plate of the strain which contain one of the parts listed in pSB3K3 .
2. Inoculate two 5ml cultures of supplemented LB Medium and antibiotic (Chloromycetin 0.1mg/ml) with single colony from the plate.
3. Cultures were grown in conical flask for 16hrs at 37℃ with shaking at 200rpm in the table concentrator.
4. Cultures were diluted 1:100 into 5ml fresh medium and grown for 3hrs at 37℃ with shaking at 200rpm in the table concentrator.
5. Then the culture was spun down and washed twice with phosphate-buffered saline (PBS, pH7.4) 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 fluorescenceand absorbance (SpectraMax+M5 microplate reader ,200ul quartz cell, Wavelengths: Excitation max: 501nm Emission max: 514nm Auto-cutoff: 515nm) every 30 minutes in the next 4hrs.