Difference between revisions of "Part:BBa K3034000"
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===Expression of CrpP===
 
===Expression of CrpP===
 
Because expression of CrpP is unsatisfying in ''E.coli'' DH5α, so we clone pelB-5D, CrpP with 6×Histag and TagRFP CDS into an expression vector (pEASY), 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 is unsatisfying in ''E.coli'' DH5α, so we clone pelB-5D, CrpP with 6×Histag and TagRFP CDS into an expression vector (pEASY), 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: CrpP expression.png|500px|thumb|center|'''Fig. 1. ''' SDS-PAGE analysis of TagRFP and CrpP from E.coli DH5α co-transformed piGEM2019-01 and piGEM2019-02 (a) and E.coli BL21 (DE3) transformed pEASY with pelB-5D, CrpP and TagRFP (b). In (a), 3,4 were the IPTG induction group, 1,2 were the control group. In (b), 1,2 were the IPTG induction group, 3,4 were the control group. The band at 25kDa is TagRFP, and the band at 17kDa is CrpP (with Histag). ]]
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[[File: CrpP expression.png|500px|thumb|center|'''Fig. 1. ''' SDS-PAGE analysis of cell extracts of engineered E.coli. In panel (a), lane 1,2: control group (E.coli DH5α (piGEM2019-01)); lane 3,4: experimental group(E.coli DH5α (piGEM2019-01 + piGEM2019-02)). In panel (b), lane 1,2: experimental group E.coli BL21 (DE3) (pEASY-crpP) (0.5 mM IPTG +); lane 3,4: control group E.coli BL21 (DE3) (pEASY-crpP) (0.5 mM IPTG -). The bands for TagRFP and CrpP (with Histag) are shown as arrows. ]]
  
 
===Characterization of CrpP function===
 
===Characterization of CrpP function===

Revision as of 08:14, 21 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 has certain value. 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 followed by a 6×Histag, the weight reaches about 15kDa.

Expression of CrpP

Because expression of CrpP is unsatisfying in E.coli DH5α, so we clone pelB-5D, CrpP with 6×Histag and TagRFP CDS into an expression vector (pEASY), 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. 1. SDS-PAGE analysis of cell extracts of engineered E.coli. In panel (a), lane 1,2: control group (E.coli DH5α (piGEM2019-01)); lane 3,4: experimental group(E.coli DH5α (piGEM2019-01 + piGEM2019-02)). In panel (b), lane 1,2: experimental group E.coli BL21 (DE3) (pEASY-crpP) (0.5 mM IPTG +); lane 3,4: control group E.coli BL21 (DE3) (pEASY-crpP) (0.5 mM IPTG -). The bands for TagRFP and CrpP (with Histag) are shown as arrows.

Characterization of CrpP function

In order to detect CrpP’s function, High Performance Liquid Chromatography (HPLC) was chosen. Although HPLC has a relatively higher sensitivity, the concentration of CIP inferred from the Km and Vmax given by Víctor M. Chávez-Jacobo et al. (2018) [1] is beyond HPLC’s limit of detection (LOD). So, we had to low down the CIP’s concentration. However, it was difficult to tell the difference between experimental group(CrpP+) and control group (CrpP-) directly through peak shape in such a low CIP concentration (＜1mg/L).

Statistics methods were introduced to help to solve this problem. Same concentration of CIP was incubated in a 1.9mL mix including 2 mM ATP, with or without cell extract that contains CrpP, at 37℃ for 30 minutes. Our experimental group (CrpP+) has 30 samples and control group (CrpP-) has 6 samples. Reaction was terminated by adding 2μL Concentrated Hydrochloric Acid. It is reasonable to reckon that the average concentration of CIP of both group has no difference in the beginning, but we can clearly notice that the experimental group’s average concentration is lower than that of control group (Fig. 2). Moreover, T-test for comparison of pooled data mean was introduced to analyze our results. T-test results indicate that in the case of a probability of error of less than 5%, there is a significant difference between the experimental group (CrpP+) and the control group (CrpP-). Thus, we can come to the conclusion that CrpP has the capability of degrading CIP.

Fig. 2. Degradation of CIP by CrpP (CrpP-: E.coli without crpP gene;CrpP+: E.coli express 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.