Team:HZAU-China/Design
Design
The core targets of our project will be implemented by engineered E. coli. It consists of three main functional modules, "Detection & Report" module, "Sterilization" module and "Repair" module. The "Detection & Report" module can detect the amount of Streptococcus mutans by sensing a signal molecule, Competence-Stimulating Peptide (CSP), as well as lead to the production of fluorescent proteins with different colors to report conditions of users' teeth. The "Repair" module can repair the enamel by producing an enamel repair protein and the "Sterilization" module can kill S. mutans by secreting a chimeric lysin. When large numbers of S. mutans are present, they can form biofilms on the teeth surface and create a low pH environment by producing acid. The enamel repair protein cannot work well in this case. Therefore, the ON and OFF states of the "Sterilization" and "Repair" module were designed based on the detection result of the amount of S. mutans, thus improving the efficiency of enamel repair. In conclusion, the engineered bacteria have three response patterns depending on the level of S. mutans.
1. Safe: No detectable S. mutans exists. Dental hygiene is good. "Detection & Report" module does not produce fluorescent proteins.
2. Solvable: S. mutans exist but the amount is low. Dental hygiene is average. "Detection & Report" module produces green fluorescent proteins. In this case, engineered bacteria will shut down the repair module temporarily and kill S. mutans first.
3. Dangerous: There is a large number of S. mutans on the teeth surface. Dental hygiene is poor. "Detection & Report" module produces green fluorescent proteins and red fluorescent proteins, which are stimulated simultaneously to produce a mixed fluorescence color close to orange. In this case, the engineered bacteria will do their best to kill S. mutans.
The three different patterns and the outline of circuits are described in Figure 1.
Figure 1. Three different response patterns and the outline of gene circuits in the engineered bacteria.
"Detection & Report" module
CSP is a quorum sensing signaling molecule of S. mutans, and its concentration is positively correlated with the amount of S. mutans. The core components of the "Detection & Report" module are two CSP-sensitive promoters from S. mutans. These promoters depend on the ComDE two-component system of S. mutans. ComD is a membrane-bound histidine kinase (HK) that is capable of autophosphorylation upon sensing CSP. The response regulator (RR) ComE then catalyzes the transfer of the phosphoryl group to an aspartate residue in its receiver domain. The phosphorylated ComE (P-ComE) will transform into an active conformation allosterically, bind to the upstream of the relevant promoters described previously and activate transcription [1]. The circuit and brief mechanism of ComDE are shown below (Figure 2).
To detect the amount of S. mutans on the teeth surface, we selected two promoters with different affinity to P-ComE. Promoter nlmC has a stronger affinity to P-ComE. It will exhibit strong transcriptional activity and express GFP and T7 RNA polymerase at a medium CSP concentration [2]. In contrast, nlmAB has a weaker affinity to P-ComE, which will show strong transcriptional activity and produce mCherry at a high CSP concentration [3]. The pattern of this module is shown below (Figure 3).
"Sterilization" module
The "Detection & Report" module will produce T7 RNAP after sensing S. mutans, which can activate the transcription of T7 promoter. In the T7 promoter expression products, ClyR will be secreted to the outside of the cell with the guide of PhoA signal peptide to kill S. mutans in the oral cavity, and TetR will temporarily shut down the repair module until the amount of S. mutans is low enough (Figure 4). ClyR, a chimeric lysin, is composed of a catalytic domain from PlyC and a cell-wall binding domain from PlySs2. ClyR has a good killing ability to some species of Streptococcus, especially to the Streptococcus mutans. ClyR can recognize and lyse the cell wall of S. mutans and break the biofilm formed by S. mutans on the teeth surface [4].
"Repair" module
The repair module is regulated by Ptet promoter. when the concentration of CSP reaches the detection threshold, TetR will be expressed to inhibit the transcription of Ptet thus shutting down the repair module (Figure 4).
The enamel repair function is mainly accomplished by LRAP, a truncated form of porcine amelogenin. It can self-assemble and serve as a framework for hydroxyapatite crystals, allowing the damaged site to reform enamel similar to its natural structure [5].
Suicide switch
We also added a suicide function to avoid the risk of the engineered bacteria entering the mouth, gastrointestinal tract, or leaking into the environment. The main components of this part are toxin MazF and promoter fhuA1. PfhuA1 is sensitive to ferrous ions. Ferrous ions can bind to Fur protein, making it an active repressor, thereby shutting down the transcription of the PfuhA1. MazF is a toxic protein with RNase activity, which can recognize the XACA sequence in ssRNA [6], hydrolyze the phosphodiester bond connecting the first adenine nucleotide, inhibit the synthesis of most proteins, and induce programmed cell death [7].
Besides, due to the high concentration of Fur protein produced on the genome of E. coli, no additional Fur protein is required to be expressed, which makes the suicide module more concise. PfhuA1 also has an appropriate sensibility to ferrous ions for our project. It would not be activated under common eutrophic culture conditions but would be activated in the oral cavity, gastrointestinal tract and natural environment where there is a lack of free ferrous ion [8]. This property allows the suicide module to confine engineered bacteria to the braces, thus preventing potential risks that can result from leakage. The pattern of this part is described in Figure 5.
Reference
[1] Lemos J A, Palmer S R, Zeng L, et al. The biology of Streptococcus mutans[J]. Gram‐Positive Pathogens, 2019: 435-448.
[2] Liu T, Xue S, Cai W, et al. ComCED signal loop precisely regulates nlmC expression in Streptococcus mutans[J]. Annals of microbiology, 2014, 64(1): 31-38.
[3] van der Ploeg J R. Regulation of bacteriocin production in Streptococcus mutans by the quorum-sensing system required for development of genetic competence[J]. Journal of bacteriology, 2005, 187(12): 3980-3989.
[4] Xu J, Yang H, Bi Y, et al. Activity of the chimeric lysin ClyR against common Gram-positive oral microbes and its anticaries efficacy in rat models[J]. Viruses, 2018, 10(7): 380.
[5] Le Norcy E, Kwak S Y, Wiedemann-Bidlack F B, et al. Leucine-rich amelogenin peptides regulate mineralization in vitro[J]. Journal of dental research, 2011, 90(9): 1091-1097.
[6] Zhang Y, Zhang J, Hoeflich K P, et al. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli[J]. Molecular cell, 2003, 12(4): 913-923.
[7] Engelberg-Kulka H, Hazan R, Amitai S. mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria[J]. Journal of cell science, 2005, 118(19): 4327-4332.
[8] Guan L, Liu Q, Li C, et al. Development of a Fur-dependent and tightly regulated expression system in Escherichia colifor toxic protein synthesis[J]. BMC biotechnology, 2013, 13(1): 25.
