Difference between revisions of "Part:BBa K4385014"
Line 3: | Line 3: | ||
<partinfo>BBa_K4385014 short</partinfo> | <partinfo>BBa_K4385014 short</partinfo> | ||
− | Lead constitutive surface display consists of the constitutive promoter J23100, the lead-inducible regulatory protein PbrR and the anchoring protein Lpp-OmpA. | + | 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=== | ===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) | ||
+ | [[Image: Pb_sensor1.png|center|frame|100px|<b>Figure 1.Pbr-PbrR-Pbc-GFP.</b>]]<br><br> | ||
+ | |||
+ | 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> | ||
<!-- --> | <!-- --> |
Revision as of 03:06, 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)
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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 938
Illegal PstI site found at 880 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1255
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 938
Illegal PstI site found at 880 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 938
Illegal PstI site found at 880 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 671