Difference between revisions of "Part:BBa K3408011"
Line 2: Line 2:
 
<partinfo>BBa_K3408011 short</partinfo>
 
<partinfo>BBa_K3408011 short</partinfo>
  
−
This composite part consists of promoter P<sub>liaG</sub> and P<sub>grac</sub>, lacI gene, CⅠ gene, GFP gene and some essential RBS and terminators. Promoter P<sub>liaG</sub> starts transcription process and its transcription production is LacI, which is regulated by IPTG. When bacteria is exposed to medium which is rich in IPTG, bacteria can intake IPTG. IPTG is combined with LacI, then LacI cannot be bound to promoter Pgrac which is specially suppressed by LacI. Therefore, CⅠ repressor protein cannot generate as downstream cⅠ gene cannot transcribe regularly.
+
This composite part consists of promoter P<sub>liaG</sub> and P<sub>grac</sub>, lacI gene, CⅠ gene, GFP gene and some essential RBS and terminators. Promoter P<sub>liaG</sub> starts transcription process and its transcription production is LacI, which is regulated by IPTG. When bacteria is exposed to medium which is rich in IPTG, bacteria can intake IPTG. IPTG is combined with LacI, then LacI cannot be bound to promoter P<sub>grac</sub> which is specially suppressed by LacI. Therefore, CⅠ repressor protein cannot generate.
  
 
As a result, P<sub>CⅠ</sub> recovers its activity and triggers the downstream transcription of GFP. By checking the expression of GFP, we could verify the feasibility of IPTG induction system, thus guaranteeing the successful culture of our engineered <i>Bacillus subtilis</i>.
 
As a result, P<sub>CⅠ</sub> recovers its activity and triggers the downstream transcription of GFP. By checking the expression of GFP, we could verify the feasibility of IPTG induction system, thus guaranteeing the successful culture of our engineered <i>Bacillus subtilis</i>.
  
  
−
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 P<sub>liaG</sub>-lacⅠ-P<sub>grac</sub>-CⅠ-P<sub>CⅠ</sub>-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 fragment to construct the expression vector P<sub>liaG</sub>-lacⅠ-P<sub>grac</sub>-CⅠ-P<sub>CⅠ</sub>-GFP.
Line 22: Line 22:
  
  
−
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 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
+
1.3.Characterization experiment
  
 
Take 2 bottles of 50ml LB liquid medium with 10μg/mL kanamycin, and inoculate the same amount of recombinant engineering bacteria.
 
Take 2 bottles of 50ml LB liquid medium with 10μg/mL kanamycin, and inoculate the same amount of recombinant engineering bacteria.
Line 36: Line 36:
  
  
−
2. Expected results
+
2. Expected results
  
 
Fluorescence can be observed in the negative control group but the test group cannot.
 
Fluorescence can be observed in the negative control group but the test group cannot.

Revision as of 03:27, 25 October 2020

PliaG-B0034-lacI-Pgrac-B0034-CⅠ-PCⅠ-GFP-B0015

This composite part consists of promoter PliaG and Pgrac, lacI gene, CⅠ gene, GFP gene and some essential RBS and terminators. Promoter PliaG starts transcription process and its transcription production is LacI, which is regulated by IPTG. When bacteria is exposed to medium which is rich in IPTG, bacteria can intake IPTG. IPTG is combined with LacI, then LacI cannot be bound to promoter Pgrac which is specially suppressed by LacI. Therefore, CⅠ repressor protein cannot generate.

As a result, PCⅠ recovers its activity and triggers the downstream transcription of GFP. 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 fragment to construct the expression vector PliaG-lacⅠ-Pgrac-CⅠ-PCⅠ-GFP.

Plasmid profile

Fig.1. The expression vector of device PliaG-lacⅠ-Pgrac-CⅠ-PCⅠ-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 10μg/mL kanamycin, and incubate them overnight at 37°C. Send transformants to biotechnology company for sequencing.


1.3.Characterization experiment

Take 2 bottles of 50ml LB liquid medium with 10μg/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 the test group cannot.

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