Antibiotic is extensively used for prevention and treatment of bacterial infection. It works well in restraining bacteria due to its complex and unique structure. However, it’s too stable to degrade, so that it can easily accumulates in the environment. With antibiotic abusing, the residue problems lead to enhancement of bacterial resistance, and become more and more serious. Even though antibiotic residues are in micrograms to nanograms levels, antibiotic residue has brought great challenges to human health and environment. Meanwhile, it’s the reason why it’s difficult to detect with conventional analysis methods, and the detection limits are too high. Apart from that, these methods’ cost is too high to be applied widely, such as HPLC. To conclude, the market urgently needs a sensitive, rapid, and efficient method of detection of antibiotic residues^[1].





       Bio-sensor is an analytical test device which uses biologically active functional unit as a biosensor, to identify target molecules through the transducer, and transform bio-chemical reaction into visual signal^[2]. However, bio-sensors also have many problems such as high detection limit, long detection time and that it’s easy to lead to false negative results^[3].

 

       Positive feedback system has been used to quantify detection, such as RT-qPCR. Because it’s more stable…In order to solve the problem mentioned above, we designed the positive feedback system as an detector which be made couple with the bio-sensor to realize trace detection of antibiotic residue.
       We take Ptet as an sensor to detect tetracycline. In the sensor, inducible promoter Ptet can identifies tetracycline or its analogues, and our sensor would transform them into the AHL signal. Then AHL is amplified through the positive feedback loop in detector, so we can utilize a mathematical model to calculate its concentration through different opening threshold. We can also test different kinds of low concentration molecular when we replace the sensor.

 

       We selected LuxI and LuxR as our positive feedback system core biobrick. The former can synthesize the N-acylhomoserine lactone (AHL) which is the key molecule of the system .It diffuses in and out of the cell membrane, and increases in concentration with cell growing. The latter codes for a protein with two functional domains containing a cytoplasmic autoinducer receptor and a DNA-binding transcriptional activator^[4].
       In our circuit, the AHL will bind to LuxR protein specifically. Then the compound can induce the Plux promoter to activate the transcription of LuxI gene and the reporter gene ^[5]. And the LuxI would produce more AHL which can bind more LuxR to activate the transcription. That would form the positive feedback loop. The positive feedback system amplifies the trace signal we have entered and transform it into a detectable fluorescent signal.

 

       Once there is Tetracycline or its analogues, the TetR protein conformation will change, so that it can control the expression of its target protein. When there is no Tetracycline in the cell, there is a specific affinity between the repressor protein “TetR” and the operator gene “TetO”, with blocking the expression of LuxI gene. In the presence of Tetracycline, the TetR protein’s conformation is changed, resulting in the separation of TetR and TetO. We used it to control the LuxI expression so that the tet signal could be convert into AHL signal.

 

       E. coli BL21 is a common strain, which is usually used to express exogenous proteins. In order to enhance its stability for emitting fluorescence , we chose E. coli BL21 as our system chassis.

 

1) Switch installation
   Considering the impact of cell growing, we insert the operon gene lacO between the promoter PLux and the LuxI, aiming to have a shorter time and a higher detection limit. We intend to open the system while the host grows steadily.
2) Background expression nutralization
   Most promoters have the constitutive transcriptional activity. And our positive feedback system may have a strong input signal because of its background expression. So AiiA gene has been select to control the background expression of our positive feedback system, which can degrade AHL.

 

[1]Nian Xiaofeng. Research Progress of Determination Methods for Tetracyclines Antibiotic Residues[J]. Chinese Journal of Veterinary Drug, 2010, 44(05):47-50.
[2]Xue Mingyue, Qin Yingfeng, Li Jian, Ye Gaojie, Zhan Zhihua. Advance Based on Signal Amplification Technology with Aptamer Biosensor[J]. Biotechnology Bulletin, 2015, 31(01):67-72.
[3]Wang Mingzhu，Xu Fei，Cao Hui，Yu Jinsong. Research progress on enzyme biosensors for detection of pesticide residue[J]. Transducer and Microsystem Technologies, 2012, 31(03):4-7.
[4]Engebrecht J, Nealson K, Silverman M. Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri. Cell, 1983, 32:773–81.
[5]Zhang Xiaobing, Fu Weiling. Research progress on bacterial quorum sensing system[J]. Chinese Journal of Nosocomiology, 2010, 20(11):1639-1642.