Difference between revisions of "Part:BBa K3408009"

Latest revision as of 02:32, 28 October 2020

P_liaG-trigger RNA-B0015-P_CⅠ-switch RNA-GFP-B0015

We used constitutive P_liaG to transcribe trigger RNA all the time. And we also used P_CⅠ as a constitutive promoter to transcribe switch RNA. If our toehold switch could work normally, GFP could be translated and expressed in the engineered bacteria.

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- trigger RNA-P_CⅠ-switch RNA-GFP.

Plasmid profile

Fig.1. The expression vector of device P_liaG- trigger RNA-P_CⅠ-switch RNA-GFP

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

Take 2 bottles of 50 ml LB liquid medium with 10 μg/mL kanamycin. 10 μg/mL kanamycin is added to the one used for the test group while the other used for the control group is not. Test group in which engineered bacteria are successfully transformed, and control group in which pWB980-DB is transformed, are inoculated by the same amount of bacteria. After culturing them for a period of time, 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 test group but not be observed in the negative control 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 239
Illegal BglII site found at 461
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1190

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K3408009 short</partinfo>
-This composite part consists of promoter P<sub>liaG</sub> and P<sub>grac</sub>, lacI gene, C&#8544; 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, C&#8544; repressor protein cannot generate as downstream cI gene cannot transcribe regularly.
+We used constitutive P<sub>liaG</sub> to transcribe trigger RNA all the time. And we also used P<sub>CⅠ</sub> as a constitutive promoter to transcribe switch RNA. If our toehold switch could work normally, GFP could be translated and expressed in the engineered bacteria.
-As a result, P<sub>C&#8544;</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>.
+. Experimental methods
+.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<sub>liaG</sub>- trigger RNA-P<sub>CⅠ</sub>-switch RNA-GFP.
+Plasmid profile
+<html>
+<img src="https://2020.igem.org/wiki/images/e/ea/T--NAU-CHINA--composite-parts4.1.png" style="width:40%"/>
+</html>
+Fig.1. The expression vector of device P<sub>liaG</sub>- trigger RNA-P<sub>CⅠ</sub>-switch RNA-GFP
+.2.Construction and screening of recombinant engineered bacteria
+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.
+.3.Characterization experiment
+Take 2 bottles of 50 ml LB liquid medium with 10 μg/mL kanamycin. 10 μg/mL kanamycin is added to the one used for the test group while the other used for the control group is not. Test group in which engineered bacteria are successfully transformed, and control group in which pWB980-DB is transformed, are inoculated by the same amount of bacteria.
+After culturing them for a period of time, use the fluorescence microscope to observe the presence of fluorescence in the test group and the control group.
+. Expected results
+Fluorescence can be observed in the test group but not be observed in the negative control group.
+<html>
+<img src="https://2020.igem.org/wiki/images/2/27/T--NAU-CHINA--composite-partsexperiment1.png" style="width:40%"/>
+</html>
+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.
 <!-- Add more about the biology of this part here