Part:BBa_K3408010

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

We designed this composite part by combining promoter 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 suicide protein—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, promoter 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_C1, the trigger RNA can successfully trigger the Toehold switch, thus leading to the expression of mazF. So Bacillus subtilis can commit suicide. While in aerobic environment, promoter P_nar 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.

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 expression vector Pnar-trigger RNA-PCⅠ-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 10μg/mL kanamycin, and incubate them overnight at 37°C. Send transformants to biotechnology company for sequencing.

1.3. Characterization experiment

①Take 2 bottles of 50ml LB liquid medium with 10μg/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

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]