Best Composite Part

Overview

This year, we aim to construct a diseases-monitoring system for automatic agriculture (AA) in which biological, physical and chemical elements can act as a whole. DAPG plays an essential role in operation of the system including serving as signal molecule or effector. Thanks to BBa_K2207013, we successfully made E.coli synthesize the DAPG and achieved our goal.

Introduction

2,4-DAPG (DAPG), full name 2,4-diacetylphloroglucinol, is a natural phenol found in specific strains of Gram-negative bacterium Pseudomonas fluorescens. This compound is found to be anti-phytopathogenic and plays an important role in the biocontrol of many plant pathogens^[1]. Meanwhile, 2,4-DAPG has also shown to induce systematic resistance in plants and enhance the plant’s resistance^[2]. What’s more, DAPG can also serve as signal molecule to construct a transcriptional switch that is similar to Tet on/off system^[3]. What’s also worth mentioning is that previous studies confirmed that DAPG almost has no harmful impact on human health under the effective concentration, and can be degraded easily in nature. ^[4]

Design

As has mentioned before, DAPG is initially found in specific strain of Pseudomonas fluorescens. And it has been demonstrated that a cluster named phl responsible for the biosynthesis of DAPG, which contains eight genes, from phlA to phlH. Further studies shows that phlABCD are the DAPG synthesis genes. ^[5][6][7]

Fig.1 2,4-DAPG synthesis pathway

We successfully cloned the phlABCD via colony PCR from Pseudomonas fluorescens 2P24. We put these four genes, phlABCD, under the control of a strong promoter which is present in the plasmid backbone we used. This backbone(BBa_K525998) was obtained from the part registry. We call this new biobrick 2,4-DAPG PhlABCD Cluster (BBa_K2207997). Upon transformation of this biobrick into BL21 E. coli cells and induced with IPTG，our engineered bacteria will synthesize DAPG.

Result

We utilize the High-performance liquid chromatography (HPLC) method to detect the biosynthesized DAPG. Luckily, we finally detected the DAPG from E.coli cells as Fig. 2. shows.

Fig.4 Results of DAPG detecting by HPLC

In this way, we successfully confirmed that this biobrick can work in E.coli.

Application Prospect

We have confirmed that the biobrick can work smoothly in E.coli, and the amount of biosynthesized DAPG is considerable. Therefore, we believe this part has promising application prospect.

DAPG can act as signal molecules to affect the structure and function of the relative protein (phlF). Therefore, we can make use this biobrick to construct a new type of transcriptional switch together with phlF protein and phlO operator. We will have other choice other than tet system. Meanwhile, it is demonstrated that the transcriptional regulation is rapid, so this set of system has strong potential to be applied to several signal transduction pathways that requires higher sensitivity.

DAPG can act as antibiotic, and be toxic to certain organisms. So we can use this biobrick to construct a type of kill switch for some organisms together with phlE(BBa_ K2207031), which is codes for an efflux pump^[8].

DAPG can act as induction factors of ISR. The plants' autoimmune mechanism can be activated under the presence of the DAPG, which will lead the plants to their prime state. Thus, the plants will have stronger resistance to the following diseases and invasion.

Reference

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[1] https://en.wikipedia.org/wiki/DAPG

[2] Rezzonico F, Zala M, Keel C, et al. Is the ability of biocontrol fluorescent pseudomonads to produce the antifungal metabolite 2,4‐diacetylphloroglucinol really synonymous with higher plant protection?[J]. New Phytologist, 2007, 173(4):861-872.

[3] Ikushima S, Boeke J D. New Orthogonal Transcriptional Switches Derived from Tet Repressor Homologues for Saccharomyces cerevisiae Regulated by 2, 4-Diacetylphloroglucinol and Other Ligands[J]. ACS Synthetic Biology, 2016.

[4] Gao Y, Chi J, eta. Effects of 2,4-diacetylphloroglucinol on two plant diseases[J]. Shandong Science, 2006, 19(4):36-39.

[5] Yang F, Cao Y. Biosynthesis of phloroglucinol compounds in microorganisms—review[J]. Applied Microbiology and Biotechnology, 2012, 93(2):487-495.

[6] Bangera M G, Thomashow L S. Characterization of a genomic locus required for synthesis of the antibiotic 2,4-diacetylphloroglucinol by the biological control agent Pseudomonas fluorescens Q2-87[J]. Molecular plant-microbe interactions : MPMI, 1996, 9(2):83.

[7] Bangera M G, Thomashow L S. Identification and Characterization of a Gene Cluster for Synthesis, of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol, from Pseudomonas fluorescens Q2-87[J]. Journal of Bacteriology, 1999, 181(10):3155.

[8] Bangera M G, Thomashow L S. Identification and Characterization of a Gene Cluster for Synthesis, of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol, from Pseudomonas fluorescens Q2-87[J]. Journal of Bacteriology, 1999, 181(10):3155.