Latest revision as of 01:35, 5 October 2013

pelB-pueB expression

Introduction

PUR degradation enzyme from Pseudomonas chlororaphis.

Protein expression

PueB induced by 2% xylose is expressed in E. coli MG1655. The expected size is 60kDa, although the actual protein band is slightly larger.

E. coli expressed PUR esterase PueB. The control used was E. coli cell with an empty plasmid

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 [http://2013.igem.org/Team:Imperial_College/Western_Blots 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 [http://2012.igem.org/Team:TU_Darmstadt/Protocols/pNP_Assay#Enzyme TU Darmstadt 2012 iGEM]

The assay was run in the [http://www.eppendorf.com/int/index.php?sitemap=2.1&action=products&contentid=1&catalognode=87236 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 PueB. The graph shows the concentration of the 4-Nitrophenol released during 10 minutes incubation of para-Nitrophenyl butyrate with PueB and Empty Vector, as well as the p-NP substrate by itself. Figure made by Imperial College London 2013 iGEM

PueB does not appear to have esterase activity for this substrate. There is a tiny increase in 4-NP concentration for PueB, 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 PueB, and Empty Vector show higher concentrations of 4-NP than the Substrate alone.

Growth Curves

Our PueB construct was tested for growth upon induction by addition of Xylose.

PueB growth assay. E. coli (MG1655) transformed with PueB BBa_K1149004 were grown with either 0% or 2% Xylose to induce PueB expression. There was no difference between no induction and induction as a t-test gave p = 0.6040 > 0.05. 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 PueB.

References:

[http://www.sciencedirect.com/science/article/pii/S0964830501000427 Cloning, nucleotide sequencing and characterization of a polyurethanase gene (pueB) from Pseudomonas chlororaphis (2001)]

[http://www.sciencedirect.com/science/article/pii/S0964830502000690 secretion and expression in E.coli]

Sequence and Features