Difference between revisions of "Part:BBa K3034000"

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	<partinfo>BBa_K3034000 short</partinfo>		<partinfo>BBa_K3034000 short</partinfo>
−	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.	+	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.
		+
		+	NCBI Reference Sequence: NG_062203.1. Prior to the DNA synthesis, we carried out the codon optimization.
		+
	<!-- Add more about the biology of this part here		<!-- Add more about the biology of this part here
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	===Molecular weight===		===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.	+	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 17kDa.
		+
		+	===Degradation of CIP by CrpP (HPLC-UV detection)===
		+	We co-transformed piGEM2019-01 and piGEM2019-02 into E.coli DH5α, and transformants were selected on LB agar plates using ampicillin and kanamycin. Overnight cultures of E. coli DH5α (piGEM2019-01 + piGEM2019-02) were diluted into 25 mL of fresh LB medium and were cultured at 37°C. When the culture was shaked for 1.5 hours, we added 1 mg/L CIP to induce the expression of CrpP, and the culture was incubated for additional 12 hours. Cells were harvested by centrifugation, and the pellets were suspended in 5mL PBS Buffer (pH = 7.4). Then the mix was treated with sonication. We used the cell extracts to degrade CIP, but 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.
		+
		+	In order to solve the problem mentioned above, statistics methods were introduced. Same concentration of CIP was incubated in both groups in a 1.9mL mix including 2 mM ATP, with or without cell extracts that contain CrpP, at 37℃ for 30 minutes. Reaction was terminated by adjusting pH to about 2 (by adding Concentrated Hydrochloric Acid). It is reasonable to reckon that the average concentration of CIP of both groups 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. 1). Moreover, T-test for comparison of pooled data mean was introduced to analyze our results. The results (P = 0.036) 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. [[File: Degradation of CIP by CrpP.png|500px|thumb|center|'''Fig. 1. ''' Degradation of CIP using cell extracts.
		+	CrpP -: E.coli DH5α (piGEM2019-01, without crpP); CrpP +: E.coli DH5α (piGEM2019-01 + piGEM2019-02, expressing CrpP enzyme) ]]
		+
		+	===Expression of CrpP===
		+	Because expression of CrpP is unsatisfying in ''E.coli'' DH5α, so we clone corresponding sequences into an expression vector (<i>pEASY</i>), 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.
		+	[[File: CrpP expression.png|700px|thumb|center|'''Fig. 2. ''' 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.  ]]
−	===Expression of CrpP-His===	+	===CrpP activity by coupled enzymatic assay===
−	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(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.	+	After CrpP was expressed in ''E.coli'' BL21 (DE3), we used a coupled enzymatic assay involving NADH oxidation to measure the activity of cell disruption solution on CIP. We incubated 2.0 mM CIP with 200ul cell disruption solution in a total volume of 1 ml of assay buffer (50 mM Tris [pH 7.6], 40 mM KCl, 10 mM MgCl2, 0.25 mg/ml NADH, 2.5 mM phosphoenolpyruvate, and 2.0 mM ATP) , 8.0 μl pyruvate kinase and 4.0 ul lactate dehydrogenase were added . The mixtures were incubated at 37°C for 3 min.
−	[[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.]]	+
−	===Characterization of CrpP function===	+	Fig. 3 presented that NADH oxidation rate of the experimental group (0.5 mM IPTG+) , the control group (0.5 mM IPTG-) and the blank group (PBS buffer). The initial values of the experimental group and the control group were the amount of NAD+ relative to the blank group at 0.5 min. When compared with the control and the blank, NADH oxidation rate was significantly increased following the induction of IPTG, indicating that CrpP expressed here can indeed degrade CIP.
−	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 ''K<sub>m</sub>'' and ''V<sub>max</sub>'' 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.	+	[[File:T-UESTC-China_CrpP_ol.png|500px|thumb|center|'''Fig. 3. ''' NADH oxidation rate comparisons between the experimental group with CrpP expression (IPTG+), the control group without CrpP expression (IPTG-) and the blank group (PBS). Data are means from three independent duplicate assays, with standard errors.]]
−	[[File: Degradation of CIP by CrpP.png|500px|thumb|center|'''Fig. 2. ''' Degradation of CIP by CrpP	+
−	(CrpP-: ''E.coli'' without crpP gene;CrpP+: ''E.coli'' express CrpP)]]	+
	===References===		===References===

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Latest revision as of 14:02, 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.

NCBI Reference Sequence: NG_062203.1. Prior to the DNA synthesis, we carried out the codon optimization.

Sequence and Features

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 17kDa.

Degradation of CIP by CrpP (HPLC-UV detection)

We co-transformed piGEM2019-01 and piGEM2019-02 into E.coli DH5α, and transformants were selected on LB agar plates using ampicillin and kanamycin. Overnight cultures of E. coli DH5α (piGEM2019-01 + piGEM2019-02) were diluted into 25 mL of fresh LB medium and were cultured at 37°C. When the culture was shaked for 1.5 hours, we added 1 mg/L CIP to induce the expression of CrpP, and the culture was incubated for additional 12 hours. Cells were harvested by centrifugation, and the pellets were suspended in 5mL PBS Buffer (pH = 7.4). Then the mix was treated with sonication. We used the cell extracts to degrade CIP, but 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.

In order to solve the problem mentioned above, statistics methods were introduced. Same concentration of CIP was incubated in both groups in a 1.9mL mix including 2 mM ATP, with or without cell extracts that contain CrpP, at 37℃ for 30 minutes. Reaction was terminated by adjusting pH to about 2 (by adding Concentrated Hydrochloric Acid). It is reasonable to reckon that the average concentration of CIP of both groups 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. 1). Moreover, T-test for comparison of pooled data mean was introduced to analyze our results. The results (P = 0.036) 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.

Expression of CrpP

Because expression of CrpP is unsatisfying in E.coli DH5α, so we clone corresponding sequences 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.

CrpP activity by coupled enzymatic assay

After CrpP was expressed in E.coli BL21 (DE3), we used a coupled enzymatic assay involving NADH oxidation to measure the activity of cell disruption solution on CIP. We incubated 2.0 mM CIP with 200ul cell disruption solution in a total volume of 1 ml of assay buffer (50 mM Tris [pH 7.6], 40 mM KCl, 10 mM MgCl2, 0.25 mg/ml NADH, 2.5 mM phosphoenolpyruvate, and 2.0 mM ATP) , 8.0 μl pyruvate kinase and 4.0 ul lactate dehydrogenase were added . The mixtures were incubated at 37°C for 3 min.

Fig. 3 presented that NADH oxidation rate of the experimental group (0.5 mM IPTG+) , the control group (0.5 mM IPTG-) and the blank group (PBS buffer). The initial values of the experimental group and the control group were the amount of NAD+ relative to the blank group at 0.5 min. When compared with the control and the blank, NADH oxidation rate was significantly increased following the induction of IPTG, indicating that CrpP expressed here can indeed degrade CIP.

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.