Team:Hong Kong-CUHK/Design
Banana Saviour Design
Introduction
Our project is inspired from the quorum sensing signalling system naturally occurring in the Xanthomonas campestris pv. campestris (Xcc) strain bacteria. The pathogenic bacteria communicate by the diffusible signalling factors (DSF) and these factors contribute to the virulence and the formation of biofilm. DSF are a group of cis-unsaturated fatty acid secreted by pathogenic bacteria. Specifically, DSF secreted by Xcc is the cis-11-methyl-dodecenoic acid. The double bond locating at the 2-position confer activity to the molecules.
In the Xcc, the responsible sensor kinases system detecting DSF in the environment are the RpfC-RpfG. After a series of auto-phosphorylation in RpfC, phosphotransfer from RpfC to RpfG occurs. This leads to the activation of phosphodiesterase domain in the RpfG, which involves in the degradation of the second messenger cyclic-di-GMP. As one of the downstream results, Clp binds to the promoters of target genes in Xcc, activating the expression of virulence gene. (Figure 1) Without the DSF induction, cyclic di-GMP will not be degraded but binds to the Clp. The binding between Clp and c-di-GMP prevents Clp from binding the gene promoters. Therefore, when pathogenic bacteria Xanthomonas campestris pv. musacearum (Xcm) in our project, exist in an environment, DSF will be secreted in the flora as signalling molecules. Our project thus aims at detecting the DSF by the RpfC-RpfG system to monitor the Xcm infection status in a banana.
Our Design
Engineered E. coli with RpfC-RpfG system
The responsible sensor kinase system detecting DSF in the environment compose of RpfC-RpfG phosphorylation relay. After a series of auto-phosphorylation in RpfC, phosphotransfer from RpfC to RpfG occurs. This leads to the activation of the phosphodiesterase domain in RpfG, which will degrade second messenger cyclic-di-GMP. As one of the downstream results, Clp binds to its target CBS I and CBS II. (Figure 1) Without the DSF induction, cyclic di-GMP will not be degraded but bind to the Clp. The binding between Clp and c-di-GMP prevents Clp from binding CBS.
Using the RpfC-RpfG system as foundation, we fused the PspF protein with Clp to enhance binding specificity and prevent expression leakage. The DNA binding domain in the C-terminus of pspF (BBa_K3071006) is replaced by Clp to construct a fusion transcription activator for our reporter construct (BBa_K3071024). This transcription activator can activate the CBSI & II-regulated pspA promoter (BBa_K3071014) upon diffusable signal factor (DSF) appearance.
pspA prometor is a sigma-54 (σ-54) regulated activator depending on promoter. sigm54-RNA holoenzyme (σ-54 RNAP) forms an inactive transcriptional initiation complex on this promoter, which can be activated in E. coli by the bacterial enhancer-binding protein PspF (BBa_K3071006). PspF transcription activation domain (pspF TAD) functions by binding to upstream activation sequences (UAS) near the promoter and contacting the promoter-bound σ-54 RNAP via DNA looping stabilized by integration host factor (IHF). Previous research has demonstrated the property of pspF-dependent and enhancer-specific transcription activation of pspA promoter.
Activation of PspA promoter leads to mRNA expression of the reporter eforRed chromoprotein as an output signal after detecting the existence of DSF.
With reference to the previous iGEM team of Dundee in 2014 (figure 2), the leakage of gene expression was found under the promoter PmanA. In their team project, Clp was targeting the PmanA. When the cell receptor RpfC detected DSF signal, the RpfC-RpfG system would be activated and thus leading to the binding of Clp to PmanA promoter. Their results, however, showed that the PmanA down-streamed GFP could still be expressed under the absence of DSF induction. It was suspected that protein such as cyclic AMP receptor protein and anaerobic regulator possessed sequence similarity, leading to the non-specific gene transcription promotion. To prevent the gene leakage, we proposed that using the fusion protein Clp-PspF instead of Clp.
Reference
[1] Zhou, Y., Asahara, H., Schneider, N., Dranchak, P., Inglese, J., & Chong, S. (2014). Engineering bacterial transcription regulation to create a synthetic in vitro two-hybrid system for protein interaction assays. Journal of the American Chemical Society, 136(40), 14031-14038.
[2] Chen, C. H., Lin, N. T., Hsiao, Y. M., Yang, C. Y., & Tseng, Y. H. (2010). Two non-consensus Clp binding sites are involved in upregulation of the gum operon involved in xanthan polysaccharide synthesis in Xanthomonas campestris pv. campestris (Xcc). Research in microbiology, 161(7), 583-589.
[3] Dworkin, J., Jovanovic, G., & Model, P. (1997). Role of upstream activation sequences and integration host factor in transcriptional activation by the constitutively active prokaryotic enhancer-binding protein PspF. Journal of molecular biology, 273(2), 377-388.