Difference between revisions of "Part:BBa K1412716"
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<partinfo>BBa_K1412716 short</partinfo>
 
<partinfo>BBa_K1412716 short</partinfo>
  
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This part is consist 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.
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This part is consist of Anderson promoter <bbpart>J23101</bbpart> and GFP generator <bbpart>BBa_E0240</bbpart>. It can be used for characterization of promoter <bbpart>J23101</bbpart>. When in backbone vector <bbpart>pSB3K3</bbpart>, the copies is low, so we need observe it carefully. While in backbone vector <bbpart>pSB1C3</bbpart>, the expression is strong, so we can observe it easily.
  
 
==='''Usage and Biology'''===
 
==='''Usage and Biology'''===
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===Protocol===
 
===Protocol===
  
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1. Transformed BBa_k1412716 into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37℃.
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1. Transformed <bbpart>BBa_k1412716</bbpart> 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.
 
2. Inoculate two 5 ml cultures of supplemented LB medium and antibiotic (Kanamycin 50 μg/ml) with single colony from the plate.
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(1)Fluorescence:
 
(1)Fluorescence:
  
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Device:  SpectraMax+M5 microplate reader, 96-well plates.
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Device:  [http://www.moleculardevices.com/systems/microplate-readers/multi-mode-readers/spectramax-m-series-multi-mode-microplate-readers SpectraMax+M5 microplate reader], 96-well plates.
  
 
Wavelengths: 501 nm excitation, 514 nm emission, Auto-cutoff: 515 nm.
 
Wavelengths: 501 nm excitation, 514 nm emission, Auto-cutoff: 515 nm.
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(2)OD600 (optical density at 600 nm):
 
(2)OD600 (optical density at 600 nm):
  
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Device: SpectraMax+M5 microplate reader, 96-well plates.
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Device: [http://www.moleculardevices.com/systems/microplate-readers/multi-mode-readers/spectramax-m-series-multi-mode-microplate-readers SpectraMax+M5 microplate reader], 96-well plates.
  
 
Wavelengths: 600 nm absorption.
 
Wavelengths: 600 nm absorption.

Revision as of 04:06, 5 September 2014

GFP generator with J23101

This part is consist 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.

Usage and Biology

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 706


Experimental data

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: [http://www.moleculardevices.com/systems/microplate-readers/multi-mode-readers/spectramax-m-series-multi-mode-microplate-readers 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: [http://www.moleculardevices.com/systems/microplate-readers/multi-mode-readers/spectramax-m-series-multi-mode-microplate-readers 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#