Part:BBa_K3408011

P_liaG-B0034-lacI-P_grac-B0034-CⅠ-P_CⅠ-GFP-B0015

This composite part consists of promoter P_liaG and P_grac, lacI gene, CⅠ gene, gfp gene and some essential RBS and terminators. P_liaG starts transcription process and its expression product is LacI, which is regulated by IPTG. LacI is the transcriptional repressor protein, which could bind to the specific site of promoter Pgrac to inhibit activity of Pgrac. IPTG can break out this inhibition by prior combination with LacI.

When our engineered bacteria is exposed to medium which is rich in IPTG, engineered bacteria can intake IPTG to trigger the expression of CⅠ repressor protein. Therefore, P_CⅠ is inhibited and GFP could not be expressed.

By checking the expression of GFP, we could verify the feasibility of IPTG induction system, thus guaranteeing the successful culture of our engineered Bacillus subtilis.

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_liaG-lacⅠ-P_grac-CⅠ-P_CⅠ-GFP.

Plasmid profile

Fig.1. The expression vector of device P_liaG-lacⅠ-P_grac-CⅠ-P_CⅠ-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 engineering bacteria.

① After culturing for 3 hours, the test group is cultured with 1 mM IPTG at 37 °C and 200 rpm for 2 hours while the IPTG is not added to control group.

② 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 negative control group but not be observed in 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.

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 1294
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2155