Difference between revisions of "Part:BBa K4385014"

Revision as of 03:13, 12 October 2022

Lead constitutive surface display

Lead constitutive surface display consists of the constitutive promoter J23100, the lead-inducible regulatory protein PbrR and the anchoring protein Lpp-OmpA.

Usage and Biology

J23100 continuously expresses PbrR for lead ion adsorption, and Lpp-OmpA is used as an anchoring protein to anchor PbrR on the surface of E. coli to construct a whole-cell biosorption device for lead.

Characterization

To verify the sensitivity of Pb2+-inducible promoter, a GFP was connected to report the expression intensity. Pbr-PbrR-Pbc-GFP was constructed to pSB1C3 (in Fig.1)

Figure 1.Pbr-PbrR-Pbc-GFP.

Different concentrations of Pb2+ were added to the reaction system, and the response of the promoter were reflected by detecting the change of fluorescence intensity. As shown in Fig. 2B, the expression of Pb2+-inducible promoter was optimal under 100 μM Pb2+ after being induced 12 hrs. Moreover, the Pb2+ inducible promoter continues to be effective after 16hours of induction, which shows our system’s stability.

Figure 2.Duration of Pb2+ induction.. (A) Digestion and gel electrophoresis of Pbr-PbrR-Pbc-GFP. (B) Fluorescence intensity of GFP under different concentration of Pb2+. The design construct was transformed into E. coli BL21(DE3). The Pb2+ was added when the OD600 value was reached 0.5. The fluorescence intensity was detected at indicated time and normalized by the OD600 value. *，P<0.05 compared to control using Student’s t test.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal SpeI site found at 938
Illegal PstI site found at 880
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1120
Illegal NheI site found at 1143
Illegal SpeI site found at 938
Illegal PstI site found at 880
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1255
23
INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 938
Illegal PstI site found at 880
25
INCOMPATIBLE WITH RFC[25]
Illegal SpeI site found at 938
Illegal PstI site found at 880
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 671

@@ Line 15: / Line 15: @@
 Different concentrations of Pb2+ were added to the reaction system, and the response of the promoter were reflected by detecting the change of fluorescence intensity. As shown in Fig. 2B, the expression of Pb2+-inducible promoter was optimal under 100 μM Pb2+ after being induced 12 hrs. Moreover, the Pb2+ inducible promoter continues to be effective after 16hours of induction, which shows our system’s stability.
-[[Image: Pb_sensor2.png|center|frame|100px|<b>Figure 2.Duration of Pb2+ induction..</b> (A) Digestion and gel electrophoresis of Pbr-PbrR-Pbc-GFP. (B) Fluorescence intensity of GFP under different concentration of Pb2+. The design construct was transformed into E. coli BL21(DE3). The Pb2+ was added when the OD600 value was reached 0.5. The fluorescence intensity was detected at indicated time and normalized by the OD600 value. *，P<0.05 compared to control using Student’s t test.]]<br><br>
+[[Image: Pb_sensor3.png|center|frame|100px|<b>Figure 2.Duration of Pb2+ induction..</b> (A) Digestion and gel electrophoresis of Pbr-PbrR-Pbc-GFP. (B) Fluorescence intensity of GFP under different concentration of Pb2+. The design construct was transformed into E. coli BL21(DE3). The Pb2+ was added when the OD600 value was reached 0.5. The fluorescence intensity was detected at indicated time and normalized by the OD600 value. *，P<0.05 compared to control using Student’s t test.]]<br><br>
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