Difference between revisions of "Part:BBa K3408010"

 
(5 intermediate revisions by 2 users not shown)
Line 4: Line 4:
 
We designed this composite part by combining P<sub>nar</sub>, trigger RNA, P<sub>CⅠ</sub>, switch RNA and <i>mazF</i>. So we could utilize it to verify the feasibility of our device, which comprises of toehold switch and toxin — MazF.  
 
We designed this composite part by combining P<sub>nar</sub>, trigger RNA, P<sub>CⅠ</sub>, switch RNA and <i>mazF</i>. So we could utilize it to verify the feasibility of our device, which comprises of toehold switch and toxin — 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, 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>CⅠ</sub>, the trigger RNA can successfully trigger the toehold switch, thus leading to the expression of <i>mazF</i>. So <i>Bacillus subtilis</i> can commit suicide. When in aerobic environment, P<sub>nar</sub> ceases to work, Toehold switch cannot be triggered. As a result, <i>mazF</i> can not be expressed. This device proves that our suicide module and toehold switch can successfully combine together.  
+
Former composite part BBa_K3408006 has demonstrated the feasibility of P<sub>nar</sub>, so when our engineered bacteria <i>Bacillus subtilis</i> is in anaerobic environment, P<sub>nar</sub> is activated by oxygen global regulator - FNR. Then downstream trigger RNA is expressed. As switch RNA is constantly expressed under the control of P<sub>CⅠ</sub>, the trigger RNA can successfully trigger the toehold switch, thus leading to the expression of <i>mazF</i>. So <i>Bacillus subtilis</i> can commit suicide. When in aerobic environment, P<sub>nar</sub> ceases to work, toehold switch cannot be triggered. As a result, <i>mazF</i> can not be expressed. This device proves that our suicide module and toehold switch can successfully combine together.  
  
  
Line 23: Line 23:
  
 
1.2.Construction and screening of recombinant engineered bacteria
 
1.2.Construction and screening of recombinant engineered bacteria
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.
+
 
 +
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.
  
  
Line 30: Line 31:
 
①Take 2 bottles of 50 ml LB liquid medium with 10 μg/mL kanamycin, and inoculate the same amount of recombinant engineered bacteria.
 
①Take 2 bottles of 50 ml LB liquid medium with 10 μg/mL kanamycin, and inoculate the same amount of recombinant engineered bacteria.
  
②After culturing engineered bacteria which have been transformed successfully for 6 hours, the test group is cultured in an anaerobic induced environment for 6 hours, and the negative control group is cultured in an aerobic environment for 6 hours.
+
②After culturing engineered bacteria which have been transformed successfully for 12 hours, the test group is cultured in an anaerobic induced environment for 6 hours, and the negative control group is cultured in an aerobic environment for 6 hours.
  
③Measure OD600 of bacteria liquid every 2 hours.
+
③Measure OD<sub>600</sub> of bacteria liquid by MicroplateReader.
  
  
Line 38: Line 39:
  
 
<html>
 
<html>
<img src="https://2020.igem.org/wiki/images/0/0f/T--NAU-CHINA--composite-partsexperiment7.png" style="width:40%"/>
+
<img src="https://static.igem.org/mediawiki/parts/1/13/T--NAU-CHINA--composite-partsexperiment6.png" style="width:40%"/>
 
</html>
 
</html>
  
Fig.2. Expected results: changes of OD600 over time
+
Fig.2. Expected results: changes of OD<sub>600</sub> over time
  
 
These results are predicted because of the lack of experiment for the COVID-19.
 
These results are predicted because of the lack of experiment for the COVID-19.

Latest revision as of 03:18, 28 October 2020

Pnar-B0034-Trigger RNA-B0015-PCⅠ-Switch RNA-mazF-B0015

We designed this composite part by combining Pnar, trigger RNA, PCⅠ, switch RNA and mazF. So we could utilize it to verify the feasibility of our device, which comprises of toehold switch and toxin — MazF.

Former composite part BBa_K3408006 has demonstrated the feasibility of Pnar, so when our engineered bacteria Bacillus subtilis is in anaerobic environment, Pnar is activated by oxygen global regulator - FNR. Then downstream trigger RNA is expressed. As switch RNA is constantly expressed under the control of PCⅠ, the trigger RNA can successfully trigger the toehold switch, thus leading to the expression of mazF. So Bacillus subtilis can commit suicide. When in aerobic environment, Pnar ceases to work, toehold switch cannot be triggered. As a result, mazF can not be expressed. This device proves that our suicide module and toehold switch can successfully combine together.


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 Pnar-trigger RNA-PCⅠ-switch RNA-mazF.

Plasmid profile

Fig.1. The expression vector of device Pnar-trigger RNA-PCⅠ-switch RNA-mazF


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

①Take 2 bottles of 50 ml LB liquid medium with 10 μg/mL kanamycin, and inoculate the same amount of recombinant engineered bacteria.

②After culturing engineered bacteria which have been transformed successfully for 12 hours, the test group is cultured in an anaerobic induced environment for 6 hours, and the negative control group is cultured in an aerobic environment for 6 hours.

③Measure OD600 of bacteria liquid by MicroplateReader.


2. Expected results

Fig.2. Expected results: changes of OD600 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
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 187
    Illegal BglII site found at 409
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]