Difference between revisions of "Part:BBa K3408009"
 
(10 intermediate revisions by 2 users not shown)
Line 2: Line 2:
 
<partinfo>BBa_K3408009 short</partinfo>
 
<partinfo>BBa_K3408009 short</partinfo>
  
−
We used constitutive promoter P<sub>liaG</sub> to transcribe trigger RNA all the time. And we also used P<sub>CⅠ</sub> as a constitutive promoter to transcribe switch RNA. If our Toehold switch could work normally, GFP could be translated and we could see green bacteria in our culture medium.
+
We used constitutive P<sub>liaG</sub> to transcribe trigger RNA all the time. And we also used P<sub>CⅠ</sub> as a constitutive promoter to transcribe switch RNA. If our toehold switch could work normally, GFP could be translated and expressed in the engineered bacteria.
−
 
+
−
 
+
  
  
 
1. Experimental methods
 
1. Experimental methods
  
−
1.1 Construction of the expression vector
+
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 expression vector PliaG- trigger RNA-PCⅠ-switch RNA-GFP.
+
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 expression vector P<sub>liaG</sub>- trigger RNA-P<sub>CⅠ</sub>-switch RNA-GFP.
  
 
Plasmid profile
 
Plasmid profile
Line 22: Line 20:
  
  
−
1.2. Construction and screening of recombinant engineered bacteria
+
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.
+
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.
  
  
−
1.3 Characterization experiment
+
1.3.Characterization experiment
  
−
Take 2 bottles of 50ml LB liquid medium with 10μg/mL kanamycin. 10μg/mL kanamycin is added to the one used for the test group while the other used for the control group is not. Test group in which engineered bacteria is successfully transformed, and control group in which pWB980-DB is transformed, are inoculated by the same amount bacteria.
+
Take 2 bottles of 50 ml LB liquid medium with 10 μg/mL kanamycin. 10 μg/mL kanamycin is added to the one used for the test group while the other used for the control group is not. Test group in which engineered bacteria are successfully transformed, and control group in which pWB980-DB is transformed, are inoculated by the same amount of bacteria.
 
After culturing them for a period of time, use the fluorescence microscope to observe the presence of fluorescence in the test group and the control group.
 
After culturing them for a period of time, use the fluorescence microscope to observe the presence of fluorescence in the test group and the control group.
  
Line 35: Line 33:
 
2. Expected results
 
2. Expected results
  
−
Fluorescence can be observed in the test group but not in the negative control group.
+
Fluorescence can be observed in the test group but not be observed in the negative control group.
  
 
<html>
 
<html>
−
<img src="https://2020.igem.org/wiki/images/d/d2/T--NAU-CHINA--composite-parts5.1.png" style="width:40%"/>
+
<img src="https://2020.igem.org/wiki/images/2/27/T--NAU-CHINA--composite-partsexperiment1.png" style="width:40%"/>
 
</html>
 
</html>
  
 
Fig.2. Expected results: different expressions of fluorescence between the control group and the test group.
 
Fig.2. Expected results: different expressions of fluorescence between the control group and the test group.
  
 +
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 02:32, 28 October 2020

PliaG-trigger RNA-B0015-PCⅠ-switch RNA-GFP-B0015

We used constitutive PliaG to transcribe trigger RNA all the time. And we also used PCⅠ as a constitutive promoter to transcribe switch RNA. If our toehold switch could work normally, GFP could be translated and 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 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 expression vector PliaG- trigger RNA-PCⅠ-switch RNA-GFP.

Plasmid profile

Fig.1. The expression vector of device PliaG- trigger RNA-PCⅠ-switch RNA-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. 10 μg/mL kanamycin is added to the one used for the test group while the other used for the control group is not. Test group in which engineered bacteria are successfully transformed, and control group in which pWB980-DB is transformed, are inoculated by the same amount of bacteria. After culturing them for a period of time, use the fluorescence microscope to observe the presence of fluorescence in the test group and the control group.


2. Expected results

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

Fig.2. Expected results: different expressions of fluorescence between the 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
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 239
    Illegal BglII site found at 461
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1190