Template:NAU-CHINA/sensing
Sensing Module
- Introduction
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Mycotoxins are compounds produced by mold fungi under moist conditions. The mycotoxin zearalenone is produced by Fusarium species. This compound has a high relative binding affinity for estrogen receptor. Binding of substances to the ligand-binding pocket of the estrogen receptor leads to its activation. The activated, ligand-bound estrogen receptor dissociates from the multi-protein complex, dimerizes,
and moves to the nucleus, where it can bind to an
estrogen response element (ERE), sequences in
the promoter regions of estrogen target genes.
Binding of the activated estrogen receptor dimer to
these promoter elements regulates the transcription of
downstream genes.Considering the sophisticated protein structure of estrogen
receptor which involves folding, processing, and modification
by organelles, we deliberate to choose yeast as our chassis. Speaking of yeasts, we naturally tend to think of GAL-inducible promoters. GAL-inducible promoters are commonly used for controlling gene expression in yeast. Unfortunately, the high cost of inducer, galactose, pose a significant barrier on its use in industrial production. In order to circumvent this obstacle, we designed an auxiliary plan which was basically using a zearalanone-based gene switch to activate GAL promoters. Because of its nanomolar sensitivity, zearalanone can fully induce reaction at a very low concentration. We sincerely believe that with this design using toxin derived from infected wheat and any other estrogen analogues, we can strongly promote the advanced synthetic biology and achieve a both sustainable and environmental-friendly
Approach
In our initial thought, we chose to place one constitutive promoter (TEF1) upstream of the gene estrogen receptor sequence and followed by three replicates of UAS (ERE) and a truncated Cyc1 promoter upstream the gene of GFP sequence. Consequently, intensity of fluorescence is dependent on the concentration of zearalenone.
In our auxiliary plan, the synthetic GAL upstream activation sequences (UAS) were synthesized from overlap extension of oligonucleotides. The gene switch constructs were generated by ligation of VP16 with an estrogen receptor ligand-binding domain (ER LBD) and a GAL4 DNA-binding domain (DBD). In this way, we have created a brand new combination of functional domain and activation domain: VP16-Gal4 DBD-ER LBD. We chose to place one constitutive promoter (TEF1) upstream of VP16-Gal4 DBD-ER LBD. ER LBD were PCR amplified from human genomic DNA, and DBD were amplified from S. cerevisiae genomic DNA. VP16 was synthesized through chemical synthesis. In our reporter plasmid, we choose to place GAL 10 promoter upstream the reporter gene sequence—GFP. Consequently, intensity of fluorescence is dependent on the concentration of the estrogen analogues—zearalenone.
Key Achievement
◎Successfully validate that under the action of a series of concentration gradients of zearalenone, the activated estrogen receptor dimer can bind to upstream activated sequence (ERE) and activate truncated Cyc 1 to transcribe GFP protein.
◎The VP16-Gal4 DBD-ER LBD Saccharomyces cerevisiae expression vector was successfully constructed and the change of green fluorescence intensity with a series of time gradients was observed under the treatment of certain concentrations of zearalenone.
Results
ZEN has a very high relative binding affinity for the estrogen receptor. Once bound and activated, the ligand-bound estrogen receptor dimerizes, moves, and binds to an estrogen response element (ERE). This results in the expression of fluorescent protein of GFP. As shown in the cellular images Fig.1A , fluorescence intensity (measured by MetaXpress image analyzer) was proportional to ZEN, ranging from 15 to 100 ng/mL. At both low and high ZEN concentrations, fluorescence intensities exhibited good linear relationships with ZEN concentrations, with correlation coefficients of 0.994. The limit of detection (LOD) calculated from Fig. 1B were 61.72 ng/mL, according to the formula: LOD = 3 s/m, where s represents the blank sample standard deviation (n = 2) and m represents the slope of the related ZEN calibration curve. These measurements were performed in triplicate to assess the reproducibility and precision of freshly fabricated yeast sensors. The relative standard deviation (RSD) of the detection results were all <5%, showing acceptable performance.
Figure 1. Quantification of ZEN by Saccharomyces cerevisiae-based sensor. (A) Fluorescence intensity spectroscopy for Saccharomyces cerevisiae treated with different doses of ZEN: 1, 2, 5, 7, 10, 15, 30, 40, 50 and 100 ng/mL. The curve of fluorescence intensity versus various concentrations of (B) ZEN. Data represent the mean ± SE of three different experiments under similar conditions.
The functionality of our auxiliary plan was confirmed by observing fluorescence from the induction of GFP on estradiol addition. The flow cytometry GFP channel histograms from the Saccharomyces cerevisiae were shown in Figure 2A. The mean fluorescence value shifted towards with the passage of time and the increase in concentration. As a result, we can conclude that the zearalanone-based gene switch can activate GAL promoters efficiently.
Figure 2. (A) Gene switch activity monitored via GFP expression and measured using flow cytometry. The GFP channel histograms are shown. (B) Gene switch activity monitored via GFP expression and measured using flow cytometry under the control of a series of time/concentration gradients. The GFP channel histograms are shown in (A).
What’s Next
◎ Due to the WiKi deadline limit, the number of experimental repetitions in our detection of zearalanone with estrogen receptors is limited. Moreover, due to time constraints, we had no time to do concentration treatment group more than 100ng / ml, through the above graph we can be see that the fluorescence intensity did not reach the platform period, which implied this system’s limit of detection (LOD) may be much lower, so more treatments and repeats are needed to increase the reliability of our final results.
◎Due to the WiKi deadline limit, we did not set repetition when we used flow cytometry to detect GFP fluorescence intensity in our auxiliary plan. From the above figure we can see the relationship between GFP intensity and time/concentration is not perfectly portrayed. Therefore, in the subsequent experiments we still need to increase the experimental repeats.
