Part:BBa_K1149004
pelB-pueB expression
Introduction
PUR degradation enzyme from Pseudomonas chlororaphis.
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.
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 results below show the concentration of 4-NP produced by the three different PUR esterases and compare them to the Empty Vector and Substrate alone as negative controls.
The above graphs clearly show that PUR Esterase EstC2 is active and cleaving p-NP. The recorded concentrations of 4-NP, in the presence of this PUR Esterase, are much greater than with the Empty Vector or the Substrate alone. Is PUR Esterase EstC2 active? Yes!
The enzymes expressed by both PueB and PulA do not appear to have esterase activity for this substrate. There is a tiny increase in 4-NP concentration for PueB, 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 PueB, PulA and Empty Vector show higher concentrations of 4-NP than the Substrate alone.
Growth Curvers
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 99
Illegal BglII site found at 1213 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 407
Illegal AgeI site found at 752
Illegal AgeI site found at 1241
Illegal AgeI site found at 1517 - 1000COMPATIBLE WITH RFC[1000]
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