Part:BBa_K4400002
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Experimental results and verification
Verification of the transformed plasmid
To verify that the gene fragment (INP and tyrosinase) is successfully expressed in the plasmid, our team completed nucleic acid gel electrophoresis to verify its presence.
As can seen by figure. 2., the marker is on the left of the gel. The second nucleic acid stain (from top to bottom) two samples to the left demonstrated the same kDa value with the marker. Thus it is verified that the modified gene expression is successful.
Construction of electrode
Functional electrodes for the biosensor were produced using the recombinant E. coliTyr cells directly adsorbed on the GC electrode. But, to confirm the construction of a functional electrode, we completed a reference experiment with the GFP gene fused with InaK-Tyr and transferred into E. coli for expression.
After the functional electrode was prepared, it was observed using fluorescence microscopy. The resulting images indicated that part of the engineered E. coli (III and IV) emitted fluorescence which was not emitted by the controlled E. coli (I and II), indicating that the engineering strains had adhered to the GC electrode to form functional E. coli-Tyr GC electrode.
Electrode response to BPA
After confirming the functional electrodes, the E. coli-Tyr GC electrode was used to investigate the presence of BPA.
We obtained the CV graph in PBS (phosphate-buffered saline) concentration of 0.1 M (and pH 7.0) with 0.1 nM of BPA, at the scan rate of 100 mV/s. This modified electrode exhibited a typical reduction current profile toward BPA (as can be seen by the yellow line on figure. 4.). On the other hand, the E. coli-psb 1a3 GC control electrode did not sense the BPA (black line on figure. 4.). These results demonstrate that the functional electrode based on the engineered strain E. coli-Tyr was successfully constructed and that it could respond to BPA.
To verify that the sensor can operate in a wide range of environments and to find the optimum response conditions in order to optimize the operating parameters to draw the calibration curve, we investigated the effects of cell loading, applied potential, pH and temperature on the performance of the biosensor.
The investigation was conducted using 50 nM of BPA.
From figures 5-8 above, the optimum working environment for the biosensor was proven to be temperature at 3 x loaded cells, -100mV, pH 7.0, and 35 °C. This environment was later utlilized in out calibration curve. Moreover, this experiment demonstrated that our biosensor is usable in a variable environment.
Calibration curve of the biosensor
After determining the optimum operating parameters for the biosensor to function, we contructed amperometry and measured amperometric response curves for successive additions of BPA standard solution to the PBS under stirring. As shown on Figure. 5, reduction current increased rapidly within the first 20 s and stabilized in 100 s. A strong linear relationship was observed between the current values and the concentration of BPA within the range of – M.
A calibration curve is contructed with calculated to be 0. 9967. The equation for the whole curve is I = − 0.04633c + 0.05368. This linear relationship demonstrated that the proposed biosensor is successful, as to give electrosignal responses to different BPA concentration.
Analysis of the tyrosinase cell-surface display system in biosensors
Tyrosinase has been widely used for BPA detection in various biosensors due to its low-cost and high activity. In these biosensors, the enzyme is usually immobilized on different chemically modified electrodes.
Although chemical modification can enhance the stability of the enzyme, the decrease in cell viability and the consequent loss in enzyme activity should be considered during the process. When compared with chemical modification, bio-modification such as microbial cell-surface display systems (as shown on Table 1), has demonstrated astonishingly low LOD values and a wide linear range.
As can be seen on table. 1., the nanographene-based tyrosinase biosensor displayed superior analytical performance over a linear range of 100-2000 , with an LOD of 33 . The gold nanoparticle modified graphene used for BPA detection also showed a good linear relationship in the concentration range of 2.5 × –3.0 μM, with an LOD of 0.001 μM.
The detection ranges of these systems were narrower than those of our biosensor, which suggests the superiority of our proposed biosensor in real environmental monitoring. The LOD of our biosensor for BPA detection was M, which is lower than that of several sensors based on chemically modified electrodes, for instance the LOD of 0.005 μM for a biosensor based on nanoflower–chitosanAuNPs/GCE, and the LOD of 0.02 mM for a biosensor based on polyglutamic acid–MWCNT– / GCE. Thus, the biosensor developed in our group is both sensitive and technically competent.
Application of the biosensor （detection in real samples)
The practical performance of the constructed biosensor in detecting the concentration of BPA in tea and juice samples was examined. To make sure our biosensor is applicable in real life, we applied our biosensor in real samples to which were added different BPA standard solutions were analyzed (as in Table 2). The BPA content in three different tea and juice samples was successfully measured using the biosensor.
The recovery rates of BPA in the tea samples were in the range of 97.78%− 98.29%, while the values in the juice samples were in the range of 98.20%− 100.32%. These results indicate that the proposed biosensor is a promising and reliable tool for accurate and quick detection of BPA in natural samples.
Conclusion
In conclusion, our experiments have demonstrated that our project is successful as to contructing a cell-surface display system of bisphenol A and that sends electric signals if BPA is detected. In our ultimate attempt is to use the biosensor to detect BPA, we received high recovery rate that demonstrates our model is able to predict BPA values with high accuracy. Moreover, proven by our experiment, biosensors the surface display system is more sensitive to BPA in the environment and thus more prefered, not to be mention its wide range compared to other biosenser configurations. Thus it is proven that our design matches our aim.
InaK+Tyrosinase
BBa_K4400002 codes for tyrosinase from Marinomonas mediterranea, a gram negative bacterium, which should facilitate expression in E. coli. Tyrosinases are native to several eukaryotic and prokaryotic species including fungi and bacteria. In its endogenous form, the tyrosinase is a part of the melanin biosynthesis pathway, converting L-Tyrosine to L-DOPA and further to dopaquinone. Melanins, dark pigments, protect the bacterial cells and spores against UV radiation, bind cytotoxic heavy metals, confer protection against oxidants, heat, enzymatic hydrolysis, antimicrobial compounds and phagocytosis (1). This part is the N-terminal domain of the ice nucleation protein. Here we established an approach to display PETase on the surface of Escherichia coli (E. coli) using N-terminal of ice nucleation protein as anchoring motif. Compared with the other anchoring motif, INP can be expressed at the cell surface of E. coli at a very high level, without affecting cell viability Bacteria cell surface display means we fix the enzyme onto the out membrane of E.coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1513
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1269
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 104
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 294
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