Team:ZJU-China/Composite Part
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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_K2207997, 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.2 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.
Composite Part BBa_K2207022
Overview
We constructed a calcium-sensitive promoter which works well in Saccharomyces cerevisiae and we chose mRFP as the report gene. This part (BBa_K2207022) can cooperate with TRPV1-Ferritin system (BBa_K2207019) to build up our Electrobiologic Interface and fulfill the signal transduction from electronic signal to gene expression. Even without TRPV1-Ferritin system, this part can be a sensitive reporter for intracellular calcium ion.
Introduction
This year, to achieve the signal transduction through medium radio frequencies (RF), we tried to construct an Electrobiologic Interface between electronic device and engineered creature. We formerly have expressed TRPV1-Ferritin in Saccharomyces cerevisiae, which can receive the medium RF signal and induce calcium influx. To complete this interface, we need a calcium-sensitive promoter. CDRE，calcineurin-dependent response element，is a segment of DNA sequence which can be regulated by specific transcription factor Crz I. This TF is activated by calcineurin in the present of calcium ion. We can construct a calcium sensitive promoter with CDRE sequence[9].
Design
We replaced the upstream activating sequence(UAS) of CYC1 promoter with 4x CDREs. We called this promoter Pcdre (BBa_K2207021) and it’s fully synthesized by UCAS through Gibson Assembly. We adapted overlap PCR to assembly it with mRFP(BBa_K2207036), which is originated from mRFP(BBa_E1010), a widely used report gene. We made a little change to BBa_E1010 to make it more suitable for expression in S. cerevisiae[9][10].
Result
We transferred this part (the backbone is pYES2.1) into S. cerevisiae BY4741 and cultured the transgenic clone in calcium-inducing medium and uninducing medium. The calcium concentration in the inducing medium is 200mM while the uninducing medium contains a relatively low concentration of calcium ion (natural medium 1x YPD). Then we detected the fluorescence of these two cultures. To eliminate the influence of the concentration of the yeast, we calculated the value of Fluorescent Intensity/OD600 to evaluate these two groups.
Fig.3 Relative fluorescent intensity of induced and uninduced groups
Fig.4 intracellular relative calcium concentration
The data shows that the relative intensity of the induced group is only 149% higher than the uninduced group. We were not content with this result, and we wondered the reason why the 200mM calcium ion cannot cause a much higher fluorescent intensity as we expected. So we tried to detect the intracellular relative calcium concentration of the two groups.
To our surprise, the difference of the intracellular calcium is not as large as we expected. When the yeast is cultured in medium containing 200mM calcium, its intracellular calcium concentration is only enhanced 8% and the basic level of the uninduced group is relative high. And these phenomena may explain the former result.
Though the expression level of the induced group does not enhance more times as we hoped, from another perspective, only the 8% increment can cause the 147% enhancement of the expression level which means this part can be a sensitive intracellular calcium reporter. Especially when considering the ability that TRPV1 can induce a more than 60% increment of intracellular calcium ion, we're sure this part will work well when combined with TRPV1-Ferritin system.
Reference
[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.
[9] Cyert M S. Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress[J]. Biochemical and biophysical research communications, 2003, 311(4): 1143-1150.
[10] Cyert M S. Genetic analysis of calmodulin and its targets in Saccharomyces cerevisiae[J]. Annual review of genetics, 2001, 35(1): 647-672.
