Difference between revisions of "Part:BBa K3408008"
 
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<partinfo>BBa_K3408008 short</partinfo>
 
<partinfo>BBa_K3408008 short</partinfo>
  
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We designed this composite part by combining promoter P<sub>nar</sub>, trigger RNA, P<sub>CⅠ</sub>, switch RNA and mazF. So we could utilize it to verify the feasibility of our device, which comprises of Toehold switch and suicide protein—mazF. Former composite part BBa_K3408006 has demonstrated the feasibility of promoter P<sub>nar</sub>, so when our engineered bacteria <i>Bacillus subtilis</i> is in anaerobic environment, promoter P<sub>nar</sub> is activated by global regulator—FNR. Then downstream trigger RNA is expressed. As switch RNA is constantly expressed under the control of promoter P<sub>C1</sub>, the trigger RNA can successfully trigger the Toehold switch, thus leading to the expression of mazF. So <i>Bacillus subtilis</i> can commit suicide. While in aerobic environment, promoter P<sub>nar</sub> ceases to work, Toehold switch cannot be triggered. As a result, mazF can not express. This device proves that our suicide module and Toehold switch can successfully combine together.  
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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 P<sub>CⅠ</sub> 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 GFP could not be expressed. But when our <i>Bacillus subtilis</i> was in an anaerobic environment, CⅠ protein could be expressed to suppress P<sub>CⅠ</sub> and we couldn't detect fluorescence intensity 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 to construct the recombinant expression vector pWB980-DB-P<sub>nar</sub>-CⅠ-P<sub>CⅠ</sub>-GFP.
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Plasmid profile
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<html>
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<img src="https://2020.igem.org/wiki/images/e/e9/T--NAU-CHINA--composite-parts3.1.png" style="width:40%"/>
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</html>
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Fig.1. The expression vector of device P<sub>nar</sub>- CⅠ-P<sub>CⅠ</sub>-GFP
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1.2.Construction and screening of recombinant engineered bacteria
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Using <i>B.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, 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 MicroplateReader 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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<html>
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<img src="https://static.igem.org/mediawiki/parts/1/13/T--NAU-CHINA--composite-partsexperiment6.png" style="width:40%"/>
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</html>
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Fig.2. Expected results: changes of fluorescence intensity under an anaerobic induced environment over time.
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These results are predicted because of the lack of experiment for the COVID-19.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 03:31, 28 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 GFP could not be expressed. But when our Bacillus subtilis was in an anaerobic environment, CⅠ protein could be expressed to suppress PCⅠ and we couldn't detect fluorescence intensity 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 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 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, the test group is cultured in an anaerobic environment, and the negative control group is cultured in an aerobic environment.

②Use the MicroplateReader 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.

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 1709