"
Team:HKU HongKong/Project
From 2012.igem.org
| Home | Team | Official Team Profile | Project | Parts Submitted to the Registry | Modeling | Notebook | Safety | Attributions |
|---|
HKU Team Project Description
Project: Inhibition of biofilm formation with engineered Escherichia coli
ABSTRACT Acyl homoserine lactones (AHLs) are important components in quorum sensing mechanism among bacteria, especially for biofilm formation. PvdQ is a protein from Pseudomonas aeruginosa with acylase function, degrading long chain AHLs. The aim of this project is to engineer a regulated PvdQ producing Escherichia coli so as to inhibit biofilm formation of Pseudomonas putida. This paves a way for living system-based biofilm inhibition research. For long term, it can be applied in industrial or environmental purpose so as to enhance biofilm clearance.
INTRODUCTION Acyl homoserine lactones are signaling molecules involved in bacterial quorum sensing. The resulting concentration of signaling molecules is dependent on the population density. pvdQ is a gene found in Pseudomonas aeruginosa, encoding PvdQ, which is an acylase that can degrade AHLs with 6 to 12 carbons in length (Joanna et al. 2007). pvdQ has been successfully cloned and expressed in E. coli (Huang et al. 2003). The role of AHLs in bacterial quorum sensing is essential (Pol et al. 2010). The novel part of this project is to relate long chains (with 6 to 12 carbons) AHLs degradation to biofilm formation.
MATERIALS AND METHODS Cloning and expressing pvdQ in E. coli pvdQ was amplified from genomic DNA of Pseudomonas Aeruginosa. A functional biobrick is constructed by combining pvdQ and some regulatory elements (such as promoter and terminator). The regulatory components are obtained from the iGEM Distribution Kit 2012. As a result, a variety of pvdQ regulatory systems can be established. Among the various regulation systems, the luxR regulation system is most concerned. luxR is a gene that can encode LuxR which binds with AHLs and upregulates the luxRp. As a result, in our biobrick model, the expression of pvdQ will be upregulated. Increase in production of pvdQ indicates an increase in acylase activity, which further degrades AHLs. The product biobrick will be a AHL-inducible acylase system. PvdQ will only be produced when AHL is present.
Testing the inhibitory effect The growth rate of monospecies biofilm of Pseudomonas putida is used to reflect the inhibitory effect of engineered Escherichia coli. This is because the major AHLs secreted by it involve 3-oxo-C12, which is a AHL that can be degraded by PvdQ and is also the major AHL produced in Pseudomonas areuginosa, the pathogenic microorganism. pvdQ expressing E. coli will be mixed with Pseudomonas putida and grow on agar plate. The reduction in biofilm formation will be assayed by crystal violet assay. The next part of the experiment is to add engineered E. coli to different phases of biofilm to validate the role of AHLs in biofilm formation.
Reference Jean J. Huang, Jong-In Han, Lian-Hui Zhang, and Jared R. Leadbetter. 2003. Utilization of Acyl-Homoserine Lactone Quorum Signals for Growth by a Soil Pseudomonad and Pseudomonas aeruginosa PAO1. Joanna Krzeslak, Mariana Wahjudi, and Wim J. Quax. 2007. Quorum quenching acylases in Pseudomonas aeruginosa.
Pol Nadal Jimenez, Gudrun Koch, Evelina Papaioannou, Mariana Wahjudi, Joanna Krzeslak, Tom Coenye, Robbert H. Cool and Wim J. Quax. 2010. Role of PvdQ in Pseudomonas aeruginosa virulence under iron-limiting conditions
Contents |
