Background and inspiration
  Environmental pollution has always been a critical issue due to the rapid population growth and the consequent industrialization [1]. Since then, monitoring and detecting pollutants is crucial [2].
  For our project, we decided to investigate methods of bisphenol A detection. Bisphenol A has been used in plenty of aspects, such as making cans, bottles, or glasses [3], since it has advantages in industrial production which are transparency, durability, anti-corrosion, etc [4].
  However, juding from the result of human practice, the majority of people have not realized how bisphenol A is harmful to our health. As a matter of fact, many health issues are due to BPA. For males, it may cause a decrease in the number of sperms, therefore affecting fertility [5]. Also, prostatic growth is related to bisphenol A, which will probably result in prostatic cancer [6]. For females, bisphenol A probably stimulates mammary hyperplasia and restricts the curative effect of chemotherapy [7]. There is also a negative influence to pregnant women. Bisphenol A is a possible cause of chromosomal defects in infants [8]. Lastly and universally, bisphenol A is known to contribute to hypertension, obesity, and cardiovascular diseases [9].
  So, the detection and monitoring of bisphenol A are important. Therefore, there is a further question, how to detect BPA?
Previous detection methods
  Traditional detection methods focus on high-performance technologies such as ultraviolet spectrometry [10] and liquid chromatography [11]. These approaches provide accurate analysis of environmental samples and have high sensitivity. However, they require costly analytical apparatus and are time-consuming, which makes them unsuitable for in-situ and rapid analysis [12]. Thus, it is desirable to develop a simple and practical analytical method for the detection of environmental pollutants [13].
Our project
  Our new detection approach is electrochemical enzyme biosensors based on engineered E. coli with a surface-display of tyrosinase, followed by absorption onto a glassy-carbon electrode.
Gene circuit
  The recombinant strain was constructed to contain the plasmid psb1a3-Tyr, which encodes the fusion protein InaK-Tyr.
Microorganisms involved in our project
Why E. coli
  E. coli is a prokaryote that does not contain a mature nucleus, and the plasmid is easy to extract. Also, they can reproduce rapidly, which is convenient in experiments. Lastly, there are no ethical issues with E. coli.
Why enzyme, and more specific, why tyrosinase
  Enzymes are relatively cost-effective and exhibit high reactivity, and selectivity [14]. However, when using chemical methods to immobilize them on electrodes, the renewability and possible loss of enzyme activity are limited.
  Tyrosinase is a kind of oxidase that can oxidize BPA to catechol. After that, catechol can still be oxidized by BPA to o-quinone. O-quinone is an electrochemically active substance that can produce electric energy to detect BPA [15].
Why ice-nucleation protein
  After collaboration with other iGEM teams, we finally chose INP as the anchor protein used in cell surface display for the following reasons. Firstly, it can be stably expressed in a large variety of species of host cells, which including E. coli. Secondly, it is uneasily to be broken down by proteases in or outside the cell. Then, it has high efficiency of displaying on the surface of the host cell. Fourthly, repeating structures can be cut without influencing functions, which can be advantageous when carrying passenger proteins with larger molecular weights. Lastly, no auxiliary proteins are needed.
  The promoter we selected is T7 promoter, which is from T7 phage. It is a strong promoter that can react specifically to T7RNA polymerase, also a sequence that initiates transcription of the T7 phage gene. T7 promoter is a kind of constitutive promoter, which is able to work continuously. This trait is useful and outstanding for our project which requires unceasingly working.
  For RBS, our team used BBa_B0034. It has a satisfactory performance and has been repeatedly verified. It can be combined with ribosomes to correctly locate to the translation initiation point, and can also control the accuracy and efficiency of mRNA translation initiation.
  BBa_B0015, the terminator we used in our project, is a double terminator combined from BBa_B0010 and BBa_B0012, which can function well and has the highest frequency of use.
Techniques involved in our project
Why cell-surface display
  Because of the drawbacks mentioned in , cell-surface display technology is chosen. By using this technology, the limitations including mass transfer and reaction rates are significantly reduced, moreover, enzyme activity can be maintained. Additionally, recent studies based on microbial cell-surface display have shown its high specificity, stability, and cost-effectiveness [16,17,18].
Why electrochemical biosensor
  It can recognize the electrical signal from the detected substance and test the concentration of the detected substance through signal strength. It has high sensitivity, accuracy, and wide applicability.
Why GC electrode
  GC is one of the most commonly used carbon materials in the laboratory due to its valuable properties such as high chemical stability [19], biocompatibility [20], and low thermal expansion coefficient [21].
System construction
  Tyrosinase display on the cell surface was achieved using the N-terminal region of the ice nucleation protein (InaK-N) [22].Then this plasmid was transformed into E. coli, to produce E. coli-Tyr. Finally, E. coli-Tyr is adsorbed to the GC electrode. Tyrosinase is a kind of oxidase that can oxidize BPA to catechol. After that, catechol can still be oxidized by BPA to o-quinone. O-quinone is an electrochemically active substance that can produce electric energy to detect BPA.
Applications
  We select 3 kinds of canned teas and juices to test the BPA concentration contained in these samples. Apart from that, for testing the accuracy of this biosensor, pure BPA solutions with different concentrations were added to the samples to calculate the BPA recovery rate.
Results and conclusions
  The biosensor measured the BPA concentrations successfully in three tea and juice samples. The recovery rate of BPA is satisfactorily high. These results reveal that our detection method is stable, reproducible, and accurate with a high recovery rate. In conclusion, these improvements provide a better solution for BPA detection in cost, accuracy, and simplification.
References
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