Difference between revisions of "Part:BBa K3408006"
 
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Used Pnar(BBa_K3408000), RBS(BBa_B0034), GFP(BBa_E0040) and terminator(BBa_B0015) to express GFP under the control of P<sub>nar</sub>. When our Bacillus subtilis was in an anerobic environment, Pnar was activated by FNR so that GFP could be expressed. But when our <i>Bacillus subtilis</i> was in an aerobic environment, P<sub>nar</sub> could be suppressed and we couldn't see GFP anymore.
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__NOTOC__
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<partinfo>BBa_K3408006 short</partinfo>
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We used P<sub>nar</sub> which was activated by FNR to trigger the expression of GFP(BBa_E0040). If there was no oxygen, then FNR could be transcribed and GFP could be expressed in the engineered bacteria.
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1. Experimental methods
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1.1. Construction of the expression vector
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The pWB980-DB is digested with enzyme EcoRI and PstI. The target fragment of the promoter, RBS, gene of green fluorescent protein (GFP) and terminator of this device are synthesized by the biotechnology company according to the known sequence. Add EcoRI and PstI restriction sites to both ends of the target fragment respectively. Connect the target fragment to the plasmid vector to construct the recombinant expression vector pWB980-DB-P<sub>nar</sub>-GFP.
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<html>
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<img src="https://2020.igem.org/wiki/images/1/18/T--NAU-CHINA--composite-parts1.1.png" style="width:40%"/>
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</html>
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Fig.1. The expression vector of device P<sub>nar</sub>-GFP
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1.2. Construction and screening of recombinant engineered bacteria
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Using <i>Bacillus subtilis</i> WB800N as the expression host, the secretion expression vector pWB980-DB was transformed by electro-transformation. Inoculate them on LB solid medium coated with 10 μg/mL kanamycin, and incubate them overnight at 37°C. Send transformants to biotechnology company for sequencing.
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1.3. Characterization experiment
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Take 2 bottles of 50 ml LB liquid medium with 10 μg/mL kanamycin, and inoculate the same amount of recombinant engineered bacteria.
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①Culture engineered bacteria which have been transformed successfully for 6 hours.
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②Culture the test group and negative control in anaerobic and aerobic environment for 6 hours respectively.
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③Use the fluorescence microscope to observe the presence of fluorescence in the test group and the negative control group.
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2. Expected results
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Fluorescence can be observed in the test group but not in the negative control group.
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<html>
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<img src="https://2020.igem.org/wiki/images/2/27/T--NAU-CHINA--composite-partsexperiment1.png" style="width:40%"/>
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</html>
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Fig.2. Expected results: different expressions of fluorescence between the negative control group and the test group.
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These results are predicted because of the lack of experiment for the COVID-19.
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<!-- -->
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3408006 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K3408006 parameters</partinfo>
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<!-- -->

Latest revision as of 02:38, 28 October 2020

Pnar-B0034-GFP-B0015

We used Pnar which was activated by FNR to trigger the expression of GFP(BBa_E0040). If there was no oxygen, then FNR could be transcribed and GFP could be expressed in the engineered bacteria.

1. Experimental methods

1.1. Construction of the expression vector

The pWB980-DB is digested with enzyme EcoRI and PstI. The target fragment of the promoter, RBS, gene of green fluorescent protein (GFP) and terminator of this device are synthesized by the biotechnology company according to the known sequence. Add EcoRI and PstI restriction sites to both ends of the target fragment respectively. Connect the target fragment to the plasmid vector to construct the recombinant expression vector pWB980-DB-Pnar-GFP.

Fig.1. The expression vector of device Pnar-GFP


1.2. Construction and screening of recombinant engineered bacteria

Using Bacillus subtilis WB800N as the expression host, the secretion expression vector pWB980-DB was transformed by electro-transformation. Inoculate them on LB solid medium coated with 10 μg/mL kanamycin, and incubate them overnight at 37°C. Send transformants to biotechnology company for sequencing.


1.3. Characterization experiment

Take 2 bottles of 50 ml LB liquid medium with 10 μg/mL kanamycin, and inoculate the same amount of recombinant engineered bacteria.

①Culture engineered bacteria which have been transformed successfully for 6 hours.

②Culture the test group and negative control in anaerobic and aerobic environment for 6 hours respectively.

③Use the fluorescence microscope to observe the presence of fluorescence in the test group and the negative control group.


2. Expected results

Fluorescence can be observed in the test group but not in the negative control group.

Fig.2. Expected results: different expressions of fluorescence between the negative control group and the test group.

These results are predicted because of the lack of experiment for the COVID-19.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 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 739