Difference between revisions of "Part:BBa K3034000"

(→‎Expression of CrpP-Histag)
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===Expression of CrpP-Histag===
 
===Expression of CrpP-Histag===
 
Because 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). 0.5mM IPTG is used for protein's expression and (over)expression is monitered by 17% SDS-PAGE.
 
Because 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). 0.5mM IPTG is used for protein's expression and (over)expression is monitered by 17% SDS-PAGE.
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[[File: SDS-PAGE of CrpP-Histag.png|500px|thumb|center|'''Fig. 1''' SDS-PAGE of CrpP-His. 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. The protein between 25kDa and 33kDa is TagRFP]]
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[[File: CrpP expression.png|500px|thumb|center|'''Fig. 1'''SDS-PAGE analysis of TagRFP and CrpP-His from E.coli DH5α co-transformed piGEM2019-01 and piGEM2019-02(a) and E.coli BL21(DE3) transformed pEASY with pelB-5D, CrpP, Histag and TagRFP(b). In (a), lane 3,4 were the IPTG induction group, lane 1,2 were the control group. In (b), lane 1,2 were the IPTG induction group, lane 3,4 were the control group.]]
  
 
===Enzyme activity of CrpP===
 
===Enzyme activity of CrpP===

Revision as of 15:25, 18 October 2019

CrpP：Ciprofloxacin-Modifying Enzyme

CrpP is a novel ciprofloxacin(CIP)-modifying enzyme which can phosphorylate 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 11 kDa.When with a 6×Histag, the weight reaches about 15kDa.

Expression of CrpP-Histag

Because 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). 0.5mM IPTG is used for protein's expression and (over)expression is monitered by 17% SDS-PAGE.

Fig. 1SDS-PAGE analysis of TagRFP and CrpP-His from E.coli DH5α co-transformed piGEM2019-01 and piGEM2019-02(a) and E.coli BL21(DE3) transformed pEASY with pelB-5D, CrpP, Histag and TagRFP(b). In (a), lane 3,4 were the IPTG induction group, lane 1,2 were the control group. In (b), lane 1,2 were the IPTG induction group, lane 3,4 were the control group.

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, Lénaïg, 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.