Composite

Part:BBa_K1149006

Designed by: Margarita Kopniczky   Group: iGEM13_Imperial_College   (2013-09-13)

pelB-pulA-flag expression

Introduction

Expression of a plastic degradation enzyme from Pseudomonas fluorescens under arabinose inducible promoter. The protein in codon optimized for expression in ecoli, is flag tagged and has an Nterminal pelB tag for secretion. Encodes polyurethane esterase, break down ester bonds in polyurethane.

PUR degradation by esterase: PUR_esterase_theoretical_pathway_2.jpg

Protein expression

Imperial PulA characterisation.png

PulA induced by 6mM arabinose is expressed in E. coli MG1655. The expected size is 48kDa, although the actual protein band is slightly larger, as shown in the figure below. The control used was E. coli cell with an empty plasmid. Some weaker bands with smaller sizes are observed, and they might be degraded PulA with his tag attached.










PUR Esterase enzyme activity

Cell lysate assay

Since the PUR Esterases were not secreted, initially the cells were lysed to obtain crude cell extracts in order to test whether the enzymes are active. The Western Blot results showed that the constructs EstC2 BBa_K1149002, PueB BBa_K1149004 and PulA BBa_K1149006 were being expressed. The cultures expressing these three constructs were grown, lysed by sonication and utilised in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. The data shows that PUR Esterase EstC2 BBa_K1149002 is definitely active.

para-Nitrophenyl butyrate (p-NP) is commonly used to indicate an enzyme’s esterase activity. The enzyme cleaves the ester bond and releases the 4-nitrophenol (4-NP), thus causing a colour change from colourless to yellow and an increased absorbance at wavelength 405 nm.



para-Nitrophenyl butyrate is cleaved by Esterases to release 4-Nitrophenol. This is accompanied by an increase in absorbance at 405 nm. Figure adapted from TU Darmstadt 2012 iGEM


The assay was run in the Eppendorf BioSpectrometer was used to automatically read the absorbance of the reaction mixture every 30 seconds. The concentration of 4-Nitrophenol produced from the reaction was calculated using the Beer-Lambert Law; the extinction coefficient of 4-NP is 18,000 M-1 cm-1 at 405 nm. [1]


The enzyme activity of PUR Esterase PulA. The graph shows the concentration of the 4-Nitrophenol released during 10 minutes incubation of para-Nitrophenyl butyrate with PulA and Empty Vector, as well as the p-NP substrate by itself.Figure made by imperial College London 2013 iGEM


PulA does not appear to have esterase activity for this substrate. There is a tiny increase in 4-NP concentration for PulA, Empty Vector and Substrate alone. This probably indicates that the substrate is slowly degrading by itself. Other constituents of the cell lysates are likely to be causing a slight increase in p-NP degradation, as PulA, and Empty Vector show higher concentrations of 4-NP than the Substrate alone.

Growth Curve

Our PulA construct was tested for growth upon induction by addition of Arabinose.

450px-PueB.png

PulA growth assay. E. coli (MG1655) transformed with PulA (BBa_K1149006) were grown with either 0 μM or 6 μM Arabinose to induce PulA expression. There was no growth inhibition as p = 0.7648 > 0.05, therefore induction did not cause significant growth decrease. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Conclusion: There is no growth inhibition caused by induction in PulA.

References:

Cloning and expression in Escherichia coli of a polyurethane-degrading enzyme from Pseudomonas fluorescens


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 804
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1099
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//function/degradation
Parameters
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