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Abstract

 

HKU’s iGEM team aims to introduce an acyl homoerine lactone (AHL)-degrading genetic system into the non-biolfilm-forming and non-virulent BL21 Escherichia coli strain. PvdQ, an enzyme naturally produced by Pseudomonas aeruginosa, is an acylase that functions to degrade long chain AHLs that bacteria like Pseudomonas putida or aeruginosa itself utilize for biofilm formation. Biofilms are population density-dependent structures formed by quorum sensing bacteria that produce and secrete auto-inducers, which signal selective gene transcription. These signaling molecules, namely the AHLs, are responsible for most bacterial pathogenicity including the opportunistic respiratory infections caused by P.aeuroginosa in immunocompromised patients.

As a step towards combating these infections, E.coli can be effectively used as a protein factory to maximize pvdQ yield in vitro or ex vivo. Our most preliminary biobrick is a constitutive promoter that drives baseline, exponential expression of pvdQ. This genetic pathway is advantageous because the pvdQ gene is constitutively transcribed regardless of environmental and endogenous factors. 

 This synthetic genetic pathway is an auto-inductive system where pvdQ protein production will specifically depend on the presence of N-dodecanoyl-L-Homoserine lactone and its coupling to the LuxR protein. Furthermore, several derivatives of the genetic system design can desirably optimize pvdQ yield. For instance, implementation of a positive feedback loop will upregulate luxR production by the simple placement of the luxR gene downstream of PluxR Larger amounts of luxR will therefore bind a greater number of AHL molecules secreted by P.aeuroginosa biofilms, thereby activating the acylase gene’s expression at a low cell density. Hence, the final biobrick produced by iGEM HKU is an AHL-inducible acylase system.

 Although the synthetic E.coli cannot be introduced into infected humans or soil and water (sources of P.aueroginosa) itself, it can be used to mass-produce pvdQ which can then be packaged into small protein-delivery bores. These structures can be stimulated to efficiently release pvdQ at the desired location, mimicking conventional drug-delivery systems. While the mechanism of pvdQ delivery will not be addressed, it can be regarded as a potential implication of HKU’s iGEM project.

 

Materials & 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 grown 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.

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