Team:ITB Indonesia/Design

Vibrio parahaemolyticus as the pathogenic bacteria in shrimp pond

Vibrio parahaemolyticus are indigenous bacteria found in all marine environment and most of the aquaculture animals such as white leg shrimp. V. parahaemolyticus is a leading cause of infectious diarrhea and enterogastritis in humans.

How did the molecular pathogenicity regulation of Vibrio parahaemolyticus?

Fig. 1. Regulation pathway of Vibrio parahaemolyticus at low and high cell density

V. parahaemolyticus uses three distinct autoinducers, Autoinducer 1 (AI-1), Autoinducer 2 (AI-2), and Cholerae Autoinducer 1 (CAI-1). Similar to V. harveyi and V. cholera, V. parahaemolyticus are synthesized this three types of autoinducer that synthesized by LuxM, LuxS, and CqsA gene. The autoinducer are binds to the receptor proteins on cell surface, LuxN, LuxP/LuxQ, and CqsS, respectively. At low cell density, the autoinducers are in low concentrations and the receptors will be autophosporylate and then transfer phosphate to the phosphorelay protein in LuxU and turn shuttles phosphate to the LuxO protein as a transcriptional factor. The phosphorylated LuxO activates the pQrr4 promoter. The overproduction of Qrr sRNAs and LuxO-P causes the feedback loops that represses the transcription of LuxO. In high cell density, the autoinducers bind to the receptors and convert the receptors to phosphatases and triggering the dephosphorylation of LuxO and LuxU, inhibits the qrr gene transcription.

Fig. 2. Regulation pathway of Vibrio parahaemolyticus at low and high cell density

V. parahaemolyticus uses quorum sensing (QS) to regulate gene expression in response to fluctuations in cell density by detecting the autoinducers. The gray fonts means the expression of relevant protein or regulatory cascades are inhibited. At low cell density, Qrr sRNAs promote Apha translation and inhibit the LuxR translation. AphA represses transcription of opaR gene. At high cell density, the absence of Qrr sRNA leads to no production of AphA and causing the translation of OpaR occurs. The production of OpaR represses the transcription of AphA and its own gene.

Our Ideas

As we have explained before, V. parahaemolyticus has unique cascade pathway to drive the expression regulation. When the cell density of bacteria is low, the cascade will stimulate the cell to produce biomass and repress the toxicity activity. But, when the cell density of bacteria is high, it will drive the expression regulation to repress the biomass production transcription factor and activate the toxicity of V. parahaemolyticus and biofilm production. This unique cascade pathway can be adopted for our system circuit to detect V. parahaemolyticus in toxic stage (high cell density). Because this cascade pathway naturally active in low cell density (without the presence of V. parahaemolyticus AI). Our circuit needs inverter module to invert the system (express reporter gene in high cell density). We used dCas9 as inverter module to invert the response of reporter gene. So to realize our system we use double plasmid system in Escherichia coli expression system.

Fig. 3. Circuits design and its regulation in high and low cell density; ((a) Cascade pathway of the circuits in the present of V. parahaemolyticus auto-inducers; (b) Cascade pathway of circuits without present of V. parahaemolyticus auto-inducers.)

How did we design the biological parts of our system?

The assembly method that we have chosen to construct our system are the Golden Gate assembly method and standard BioBricks assembly method. We have chosen this technology for several reasons: This technology uses type IIS restriction enzymes in order to cut all the parts and these parts will be used to build these genetic circuits. These enzymes are a group of endonucleases that recognize specific asymmetric double stranded DNA sequences and cleave outside of their recognition sequence. The result of this cleavage will leave short single stranded overhangs with non-specific sequences. This phenomenon allows us to define the overhangs cleavage sequence of each part to enabling assembly with multiple DNA parts specifically. Our actuator module and inverter module are consist of very large number of DNA base pairs. Synthesis of DNA base pairs in large numbers is very difficult because can cause error when it is synthesized. Golden Gate assembly allow us to assemblies the multiple of gene parts in one reaction steps. So this method allows us to assemble DNA circuits easily and quickly. Apart from using the Golden Gate Assembly, we also used standard BioBrick Assembly that utilize XbaI restriction site to construct pdCas-GFP composite part (Fig. 4).

Fig. 4. Construction scheme of pdCas9-GFP from their basic composite parts using XbaI restriction enzyme

The Golden Gate Assembly

Golden Gate Assembly is a multipartite assembly system based that used the type IIS restriction enzymes. These enzymes digest DNA at a defined distance few nucleotides away from its recognition site, not requiring any specific sequence in the actual cleavage site, and often leaving a short overhang. This feature makes this technology extremely useful in seamless cloning strategies: by carefully positioning recognition and digestion sites in opposite directions in entry and destination vectors, it is possible to design and obtain multipartite assemblies in one step reaction. Since there are no sequence requirements in the cleavage sites, these can be user defined, and therefore accommodated to serve as assembly boundaries for standard DNA parts. Following this rationale, we initially considered three categories of basic composite parts that consist of promoters, coding sequences, and terminators. This method is used for assembly the actuator module that consist of LuxO, LuxU, and LuxN. All that basic composite parts are cloned as BsaI fragments in entry clones. The inclusion in a category is defined by the flanking BsaI digestion sites. A schematic view of a standardized multipartite assembly of transcriptional unit is depicted in Fig. 5. To facilitate the interpretation, we gave a label to each 4 bp cleavage site that produced by appropriate enzymes and its ligation visualization. Vectors that contain basic composite parts are obtained from custom gene synthesis that synthesized by IDT with appropriate antibiotic marker. That vectors are designed with BsaI recognition site and specific overhang recognition site. These basic composite parts are ligate with pSB1A3 back bond plasmid that has been double digested by EcoRI and XbaI. The circuit construction that has been successfully assembled is then cloned into Escherichia coli and selected in appropriate antibiotic.

Fig. 5. Construction scheme of pSB1A3-LuxOUN from their basic composite parts using Golden Gate Assembly grammar

How it Works

Our system consists of two expression modules, actuator modules and inverter modules. The actuator modules are the series of genes that function to activate the pQrr4 short promoter. The actuator module consists of LuxN, LuxU, LuxO, pQrr4-gRNA-115 genes. LuxN is a gene that expresses the LuxN protein that functions to interact with the specific V. parahaemolyticus autoinducer. When the cell density of V. parahaemolyticus is low, autoinducer will not produced and the circuits will be off (not express reporter gene). The LuxN protein does not interact with the V. parahaemolyticus autoinducer, the LuxN protein will phosphorylate the LuxU protein that expressed by the LuxU gene. The phosphorylated LuxU protein will further phosphorylate the LuxO protein that expressed by the LuxO gene. Furthermore, the phosphorylated LuxO protein will activates pQrr4 short promoter which will express a single guide RNA (gRNA-115) to form the gRNA-dCas9 complex with the dCas9 protein expressed by the inverter vector. The inverter vector consists of the dCas9 gene and GFP as the reporter gene promoted by constitutive promoter BBa_J23115 obtained from the Anderson promoter collection. The gRNA-dCas9 complex that is formed will repress the BBa_J23115 promoter to suppress GFP expression when the presence of V. parahaemolyticus is low. Previously when the density of V. parahaemolyticus bacteria is high, the LuxN-LuxU-LuxO protein will be dephosphorylated so that gRNA is not produced and GFP protein can be expressed (see animation 1).

Animation 1. Engineered Escherichia coli system pathway animation

References
  • Holowko, M. B., Wang, H., Jayaraman, P., Poh, C. L., (2016). Biosensing Vibrio cholerae with Genetically Engineered Escherichia coli. ACS Synth. Biol. 2016, 5, 1275-1283
  • Makino K, Oshima K, Kurokawa K, Yokoyama K, Uda T, Tagomori K, Iijima Y, Najima M, Nakano M, Yamashita A, Kubota Y, Kimura S, Yasunaga T, Honda T, Shinagawa H, Hattori M, Iida T. 2003. Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae. DOI: 10.1016/S0140-6736(03)12659-1
  • Potapov, V., Ong, J. L., Kucera, R. B., Langhorst, B. W., Bilotti, K., Pryor, J. M., Cantor, E. J., Canton, B., Knight, T. F., Jr, T. C. E., Lohman, G. J. S., (2018). Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synth. Biol. 2018, 7, 2665-2674
  • Sarrion-Perdigones, A., Falconi, E., E., Zandalinas, S., I., Jua´rez, P., Ferna´ndez-del-Carmen, A., Granell, A., Orzaez, D., (2011). GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLoS ONE. 6(7): e21622
  • Zhang Y, Qiu Y, Tan Y, Guo Z, Yang R, et al. (2012) Transcriptional Regulation of opaR, qrr2–4 and aphA by the Master Quorum-Sensing Regulator OpaR in Vibrio parahaemolyticus. PLoS ONE 7(4): e34622. doi:10.1371/journal.pone.0034622
  • Zhou, D., Yan, X. , Qu, F., Wang, L., Zhang, Y ., Hou, J. , Hu,Y. , Li, J. , ojie Xin a, Jingfu Qiu, S. , Yang, R. , Mao, P. 2013.Quorum sensing modulates transcription of cpsQ-mfpABC and mfpABC in Vibrio parahaemolyticus. International Journal of Food Microbiology 166 page 458-463