Difference between revisions of "Part:BBa K3034000"

Revision as of 15:27, 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 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.

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

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.

@@ Line 17: / Line 17: @@
 ===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 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.
 [[File: SDS-PAGE of CrpP-Histag.png|500px|thumb|center|'''Fig. 1'''SDS-PAGE of CrpP-Histag. Lane 1: Protein ladder; Lane 2、3、5 and 6:  experimental group(0.5mM IPTG is added); Lane 3、4、7 and 8: control group(without 0.5mM IPTG)]]