Part:BBa_K3408010

P_nar-B0034-Trigger RNA-B0015-P_CⅠ-Switch RNA-mazF-B0015

We designed this composite part by combining P_nar, trigger RNA, P_CⅠ, 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 promoter P_nar, so when our engineered bacteria Bacillus subtilis is in anaerobic environment, P_nar is activated by global regulator—FNR. Then downstream trigger RNA is expressed. As switch RNA is constantly expressed under the control of promoter P_CⅠ, 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, P_nar 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 P_nar-trigger RNA-P_CⅠ-switch RNA-mazF.

Plasmid profile

Fig.1. The expression vector of device P_nar-trigger RNA-P_CⅠ-switch RNA-mazF

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

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.

③Measure OD600 of bacteria liquid every 2 hours.

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]