Difference between revisions of "Part:BBa K3777024"

Latest revision as of 02:56, 22 October 2021

KB2-tetR-T7(tetO)-3WJdB-Ptet-sgRNA

A biosensor device for better tetracycline detection.

Usage and Biology
Compared to our another part TetR-tetM-T7(tetO)-3WJdB(BBa_K3777014),it adds a binding site which can be regonized by dcas9 proteins and sgRNA.(Fig 1)
When tet was absent, TetR would bind to the inducible promoter（PI）and prevent RNA polymerase from initiating transcription, thus repressing the expression of reporter gene. If tet was present, TetR would no longer able to bind to the promoter, resulting in the expression of reporter gene. But KB2 will bind to 3WJDB leading to lower fluorescent .
We expressed this circuit in the E. coli BL21(DE3) cells for tetracycline detection. Thus we could roughly deduce the concentration of the antibiotics in the sample according to the fluorescence intensity.
Fig.1 Schematic overview of the genetic circuit.
Verifying the strand replacement reaction. Promoter J23104 is much stronger than J23110. TetR-T7(tetO)-3WJdB has no KB2, so it is set as a negative control.
The result of this experiment proved that it’s valid to repress the fluorescence in vivo, which strongly support the feasibility of the ‘improved circuit’.

Reference：Alam Khalid K,Tawiah Kwaku D,Lichte Matthew F,Porciani David,Burke Donald H. A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo.[J]. ACS synthetic biology,2017,6(9)
Sequence and Features Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 260
Illegal NheI site found at 283
Illegal NheI site found at 1262
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 121
Illegal XhoI site found at 1388
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 8: / Line 8: @@
 <b><font size="3">Usage and Biology</font></b>
 <br>Compared to our another part TetR-tetM-T7(tetO)-3WJdB(BBa_K3777014),it adds a binding site which can be regonized by dcas9 proteins and sgRNA.(Fig 1)
-<br>When tet was absent, TetR would bind to the inducible promoter（PI）and prevent RNA polymerase from initiating transcription, thus repressing the expression of reporter gene. If tet was present, TetR would no longer able to bind to the promoter, resulting in the expression of reporter gene. But BS2 will be regonized and cut by dca9 proteins and
+<br>When tet was absent, TetR would bind to the inducible promoter（PI）and prevent RNA polymerase from initiating transcription, thus repressing the expression of reporter gene. If tet was present, TetR would no longer able to bind to the promoter, resulting in the expression of reporter gene. But KB2 will bind to 3WJDB leading to lower fluorescent .
-sgRNA leading to lower fluorescence expression.
 <br>We expressed this circuit in the <i>E. coli </i> BL21(DE3) cells for tetracycline detection. Thus we could roughly deduce the concentration of the antibiotics in the sample according to the fluorescence intensity.
 https://static.igem.org/mediawiki/parts/thumb/1/1e/KB2-tetR-T7%28tetO%29-3WJdB-Ptet-sgRNA.PNG/799px-KB2-tetR-T7%28tetO%29-3WJdB-Ptet-sgRNA.PNG
 <br>                           Fig.1  Schematic overview of the genetic circuit.
-<br><b><font size="3">Results</font></b>
+https://static.igem.org/mediawiki/parts/thumb/3/30/T--OUC-China--result--V.png/800px-T--OUC-China--result--V.png
-<br>To verify the functionality of the biosensor, we performed a plate-reader experiment and measured optical density and fluorescence intensity every hour. We observed a correlation between concentration of antibiotics in the sample and intensity of fluorescent signal.
+<br>Verifying the strand replacement reaction. Promoter J23104 is much stronger than J23110. TetR-T7(tetO)-3WJdB has no KB2, so it is set as a negative control.
-https://static.igem.org/mediawiki/parts/3/3f/CRISPRi.PNG
+<br>The result of this experiment proved that it’s valid to repress the fluorescence in vivo, which strongly support the feasibility of the ‘improved circuit’.
-<br>The figure shows that our cuircuit can recognize different concentrations of tetracycline. What is more, CRISPRi can really inhibit our fluorescence expression
 <br>
 <br>Reference：Alam Khalid K,Tawiah Kwaku D,Lichte Matthew F,Porciani David,Burke Donald H. A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo.[J]. ACS synthetic biology,2017,6(9)