Team:iBowu-China/Design
After the preliminary ideas and plans were drawn up, we encountered several challenges, mainly including three aspects:
- We need to find key gene regulation mechanisms that can be used for the purpose of disease detection and control.
- We need to develop an approach that is no biosafety risks.
- We need to turn our system into a final product that can be consumed and used by the public, directly serving the agricultural production.
In order to address the challenges, we propose the following solutions:
- We designed biosensing and biocontrol gene circuits based on quorum sensing and antimicrobial peptide.
- We introduced cell-free synthesis into our project.
- We developed a toolkit, which can detect and prevent soft rot.
Quorum sensing (QS) is a cell-cell communication system that enables bacteria sense and respond to cell population density as well as changes by gene regulation. [1][2] As one example, quorum sensing enables bacteria to restrict the expression of specific genes in high cell population density, which is critical for bacterial life. Many species of bacteria use quorum sensing to coordinate gene expression. [3]
Acyl-homoserine lactones (AHLs) are the quorum-sensing signal molecules used to coordinate the population behaviors in gram-negative bacteria. In V. fischeri, AHLs produced by LuxI will bind with LuxR and then activate downstream gene. [4][5]
Quorum-sensing AHL autoinducer of Erwinia Carotovora is N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), and its regulatory protein is ExpR, which is the homologous protein of LuxR (V. fischeri). [6][7]
OHHL
For the termination of the disease, the circuit will either produce AiiA or Antimicrobial Peptides. AiiA is a hydrolase enzyme that will hydrolyze the AHL molecules on the surface of the affected plants, terminating the virulence of the bacteria if reached a threshold number. [4][8][9] Antimicrobial Peptides (AMP) are peptides that target the lipopolysaccharide layer of the bacteria. Electrostatic adsorption occurs between the positive surface charge and the negative charge on the surface of antimicrobial peptide and bacterial membrane. Then, the hydrophobic end of the peptide is inserted into the inner membrane, damaging the membrane structure and interfering with the osmotic pressure inside and outside the cell, resulting in the final death of the pathogen cell. [10]
We designed two gene circuits based on quorum sensing and antimicrobial peptide. One is to detect OHHL (quorum sensing signal molecular) of E.carotovora; the other is to express the hydrolase aiiA and the antibacterial peptides which could degrade OHHL and generally kill bacteria separately.
To mitigate the biological safety concerns of the entire project, we have opted to produce the gene circuits with cell free technology to avoid any unwanted leakage of hazardous biological materials. With the physical removal of the cell wall, the cell free system is easier to engineer and transport when compared with living cells. The system no longer requires constant maintenance and can be utilized with short notice. [11]
By purifying the cell extract, we created a cell free system which parts and functions we can alter easily (two gene circuits). This ease of alteration has allowed us to create multiple gene circuits and test their respective effectiveness, ensuring the quality of our end product. When dried and extracted onto testing strips, the system can be transported cheaply over long distances and utilized by persons who have very little to no knowledge of Synthetic Biology.
Our primary concern is: how do we translate our design into a toolkit that is accessible and widely used by the general public. So we made the following improvements: in our detection device, the cell-free systems coupled with synthetic networks are embedded into paper discs by freeze-drying, which are able to be distributed and store at room temperature. [12] The samples will be added for hydration when we used this paper for detection.
1. Bassler B L. How Bacteria Talk to Each Other: Regulation of Gene Expression by Quorum Sensing[J]. Current Opinion in Microbiology, 1999, 2(6):582-587.
2. Fuqua C, Parsek M R, Greenberg E P. REGULATION OF GENE EXPRESSION BY CELL-TO-CELL COMMUNICATION: Acyl-Homoserine Lactone Quorum Sensing[J]. Annual Review of Genetics, 2001, 35(1):439-468.
3. Bodman S B V, Bauer W D, Coplin D L. Quorum Sensing in Plant-Pathogenic Bacteria[J]. Annual Review of Phytopathology, 2003, 41(1):455-482.
4. Dong Y H, Xu J L, Li X Z, et al. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora[J]. Proceedings of the National Academy of Sciences, 2000, 97(7):3526-3531.
5. Dong Y H, Wang L H, Xu J L, et al. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase[J]. Nature, 2001, 411(6839):813-817.
6. Bainton N J, Stead P, Chhabra S R, et al. N-(3-Oxohexanoyl)-L-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora[J]. Biochemical Journal, 1993, 288(3):997-1004.
7. Welch M, Todd D E, Whitehead N A, et al. N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia[J]. EMBO (European Molecular Biology Organization) Journal, 2000, 19(4):631-641.
8. Liu C F, Liu D, Momb J, et al. A Phenylalanine Clamp Controls Substrate Specificity in the Quorum-Quenching Metallo-γ-lactonase from\r, Bacillus thuringiensis[J]. Biochemistry, 2013, 52(9):1603-1610.
9. Liu C F, Liu D, Momb J, et al. A distal phenylalanine clamp in a hydrophobic channel controls the substrate specificity in the quorum-quenching metallo-γ-lactonase (AiiA) from Bacillus thuringiensis[J]. Biochemistry, 2013, 52.
10. Zeitler B, Diaz A H, Dangel A, et al. De-Novo Design of Antimicrobial Peptides for Plant Protection[J]. PLOS ONE, 2013, 8.
11. Carlson E D, Gan R, Hodgman C E, et al. Cell-free protein synthesis: Applications come of age[J]. Biotechnology Advances, 2012, 30(5):1185---1194.
12. Pardee K, Green A, Ferrante T, et al. Paper-Based Synthetic Gene Networks[J]. Cell, 2014, 159(4):940-954.