Difference between revisions of "Part:BBa K3034000"

(→‎Expression of CrpP-Histag)
(→‎Expression of CrpP-Histag)
Line 17: Line 17:
  
 
===Expression of CrpP-Histag===
 
===Expression of CrpP-Histag===
−
Expression of CrpP-Histag is unsatisfying in E.coli ''DH5α'', so we clone pelB-5D, CrpP and 6×Histag CDS into an expression vector, then the vector is transformed into E.coli ''BL21(DE3)''. We use IPTG to induce its overexpression and SDS-PAGE is used to detect target protein's expression.
+
Expression of CrpP-Histag is unsatisfying in E.coli ''DH5α'', so we clone pelB-5D, CrpP with 6×Histag and TagRFP CDS into an expression vector, then the vector is transformed into E.coli ''BL21(DE3)''. We use IPTG to induce its overexpression and SDS-PAGE is used to detect target protein's expression.
−
[[File: SDS-PAGE of CrpP-Histag.png|500px|thumb|center|'''Fig. 1''' SDS-PAGE of CrpP-Histag. M: Protein ladder; Lane 2、3: experimental group 1 (0.5mM IPTG is added); Lane 3、4: control group 1 (without 0.5mM IPTG); Lane 5、6: experimental group 2; Lane 7、8: control group 2]]
+
[[File: SDS-PAGE of CrpP-Histag.png|500px|thumb|center|'''Fig. 1''' SDS-PAGE of CrpP-Histag. M: Protein ladder; Lane 2、3: experimental group 1 (0.5mM IPTG is added); Lane 3、4: control group 1 (without 0.5mM IPTG); Lane 5、6: experimental group 2; Lane 7、8: control group 2. Protein between 25kDa and 33kDa is TagRFP]]
  
 
===Enzyme activity of CrpP===
 
===Enzyme activity of CrpP===

Revision as of 15:37, 11 October 2019

CrpP：Ciprofloxacin-Modifying Enzyme

CrpP is a novel ciprofloxacin-modifying enzyme which can phosphorylate ciprofloxacin(CIP). Then the phosphorylated CIP goes through multiple steps of degradation spontaneously and produces 1,4-dihydroquinoline finally[1]. 1,4-dihydroquinoline is used as a kind of new carriers for specific brain delivery [2], so we can infer that it's nontoxic. This part is responsible for degradation of ciprofloxacin in our project.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

Molecular weight

This gene codes for a protein of 65 amino acids with a molecular mass about 15 kDa.

Expression of CrpP-Histag

Expression of CrpP-Histag is unsatisfying in E.coli DH5α, so we clone pelB-5D, CrpP with 6×Histag and TagRFP CDS into an expression vector, then the vector is transformed into E.coli BL21(DE3). We use IPTG to induce its overexpression and SDS-PAGE is used to detect target protein's expression.

Fig. 1 SDS-PAGE of CrpP-Histag. M: Protein ladder; Lane 2、3: experimental group 1 (0.5mM IPTG is added); Lane 3、4: control group 1 (without 0.5mM IPTG); Lane 5、6: experimental group 2; Lane 7、8: control group 2. Protein between 25kDa and 33kDa is TagRFP

Enzyme activity of CrpP

References

[1]Chávez-Jacobo, Víctor M., et al. CrpP is a novel ciprofloxacin-modifying enzyme encoded by the Pseudomonas aeruginosa pUM505 plasmid. Antimicrobial agents and chemotherapy 62.6 (2018): e02629-17.

[2]Foucout, et al. Synthesis, radiosynthesis and biological evaluation of 1, 4-dihydroquinoline derivatives as new carriers for specific brain delivery. Organic & biomolecular chemistry 7.18 (2009): 3666-3673.