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 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..  
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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?''' ==
 
== '''Why we build this BioBrick?''' ==
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===Protocol===
 
===Protocol===
  
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1. Streak a plate of the strain which contain one of the parts listed in pSB3K3 .
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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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2. Inoculate two 5ml cultures of supplemented LB Medium and antibiotic (Chloromycetin 0.1mg/ml) with single colony from the plate.
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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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3. Cultures were grown in conical flask for 16hrs at 37℃ with shaking at 200rpm in the table concentrator.
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3. Cultures were grown in conical flask for 16 hrs at 37℃ with shaking at 200 rpm in the table concentrator.
  
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4. Cultures were diluted 1:100 into 5ml fresh medium and grown for 3hrs at 37℃ with shaking at 200rpm in the table concentrator.
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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.
  
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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.
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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.
 
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.
  
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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.
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7. Measure the fluorescence and absorbance:
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(1)Fluorescence:
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Device:  SpectraMax+M5 microplate reader, 96-well plates.
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Wavelengths: 501 nm excitation, 514 nm emission, Auto-cutoff: 515 nm.
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(2)OD600 (optical density at 600 nm):
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Device: SpectraMax+M5 microplate reader, 96-well plates.
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Wavelengths: 600 nm absorption.
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8. Measure every 30 minutes in the next 4 hrs.
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===References===
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[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.
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----
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<I><B>More information, click here: http://2014.igem.org/Team:XMU-China#

Revision as of 05:10, 4 September 2014

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


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


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#