Difference between revisions of "Part:BBa K3408008"
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Used P<sub>nar</sub>(BBa_K3408000), RBS(BBa_B0034), optimized CⅠ(BBa_K3408004), terminator(BBa_B0015), P<sub>CⅠ</sub>(BBa_R0051), RBS(BBa_B0034) and GFP(BBa_E0040) to express GFP when PCⅠ was activated. When our <i>Bacillus subtilis</i> was in an aerobic environment, P<sub>nar</sub> was suppressed without FNR and no CⅠ protein could be expressed so that P<sub>CⅠ</sub> could be activated and we could see GFP expressed. But when our <i>Bacillus subtilis</i> was in an anerobic environment, CⅠ protein could be expressed to suppress P<sub>CⅠ</sub> and we couldn't see GFP anymore.   
 
Used P<sub>nar</sub>(BBa_K3408000), RBS(BBa_B0034), optimized CⅠ(BBa_K3408004), terminator(BBa_B0015), P<sub>CⅠ</sub>(BBa_R0051), RBS(BBa_B0034) and GFP(BBa_E0040) to express GFP when PCⅠ was activated. When our <i>Bacillus subtilis</i> was in an aerobic environment, P<sub>nar</sub> was suppressed without FNR and no CⅠ protein could be expressed so that P<sub>CⅠ</sub> could be activated and we could see GFP expressed. But when our <i>Bacillus subtilis</i> was in an anerobic environment, CⅠ protein could be expressed to suppress P<sub>CⅠ</sub> and we couldn't see GFP anymore.   
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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 phytase and terminator of this device are synthesized by the biotechnology company with 6×His tags added. Add EcoRI and PstI restriction sites to both ends of the target fragment respectively. Connect the target fragment to the plasmid vector fragment to construct the recombinant expression vector pWB980-DB-P<sub>nar</sub>-CⅠ-P<sub>CⅠ</sub>-GFP.
  
 
Plasmid profile
 
Plasmid profile
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https://2020.igem.org/wiki/images/e/e9/T--NAU-CHINA--composite-parts3.1.png
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<img src="https://2020.igem.org/wiki/images/1/18/T--NAU-CHINA--composite-parts1.3.png" style="width:40%"/>
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Fig.1. The expression vector of device P<sub>nar</sub>- CⅠ-PCⅠ-GFP
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1.2.Construction and screening of recombinant engineered bacteria
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Using B. 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 10μg/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 50ml LB liquid medium with 10μg/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, the test group is cultured in an anaerobic environment, and the negative control group is cultured in an aerobic environment.
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②Use the microplate reader to observe the presence of fluorescence in the test group and the control group at 0 min, 10 min, 20 min, 30 min, 40 min, 60 min, 80 min, and 120 min.
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2.Expected results
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The test group: the fluorescence intensity gradually decreases
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The control group: the fluorescence intensity remains unchanged
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Fig.2. Expected results: changes of fluorescence intensity under an anaerobic induced environment over time.
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<!-- Add more about the biology of this part here

Revision as of 16:58, 18 October 2020

Pnar-B0034-CⅠ-B0015-PCⅠ-B0034-GFP-B0015

Used Pnar(BBa_K3408000), RBS(BBa_B0034), optimized CⅠ(BBa_K3408004), terminator(BBa_B0015), PCⅠ(BBa_R0051), RBS(BBa_B0034) and GFP(BBa_E0040) to express GFP when PCⅠ was activated. When our Bacillus subtilis was in an aerobic environment, Pnar was suppressed without FNR and no CⅠ protein could be expressed so that PCⅠ could be activated and we could see GFP expressed. But when our Bacillus subtilis was in an anerobic environment, CⅠ protein could be expressed to suppress PCⅠ and we couldn't see GFP anymore.


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 phytase and terminator of this device are synthesized by the biotechnology company with 6×His tags added. Add EcoRI and PstI restriction sites to both ends of the target fragment respectively. Connect the target fragment to the plasmid vector fragment to construct the recombinant expression vector pWB980-DB-Pnar-CⅠ-PCⅠ-GFP.

Plasmid profile

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


1.2.Construction and screening of recombinant engineered bacteria

Using B. 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 10μg/mL kanamycin, and incubate them overnight at 37°C. Send transformants to biotechnology company for sequencing.


1.3 Characterization experiment

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

①Culture engineered bacteria which have been transformed successfully for 6 hours, the test group is cultured in an anaerobic environment, and the negative control group is cultured in an aerobic environment.

②Use the microplate reader to observe the presence of fluorescence in the test group and the control group at 0 min, 10 min, 20 min, 30 min, 40 min, 60 min, 80 min, and 120 min.


2.Expected results

The test group: the fluorescence intensity gradually decreases

The control group: the fluorescence intensity remains unchanged


Fig.2. Expected results: changes of fluorescence intensity under an anaerobic induced environment over time.


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 1709