Contributions to iGEM Registry of Standard Biological Parts
Lubbock_TTU's contribution to the Registry consists of the creation and documentation of four parts and the whole biosensor. These four parts are derived from Vibrio cholerae ’s chitin utilization program and expressed in E. coli to serve as players in our chitin detection system. We want to further explore and collect data on these parts and the system so that other iGEM teams in the future can potentially utilize them for their projects. The one-component system is still an active area of research, with much more to discover mechanistically, giving other teams the potential to conduct research in this as well. Furthermore, our ChiSPY system can potentially contribute to other iGEM team’s future projects by serving as an E.Coli based chassis detection system.
Pchb is a promoter codon-optimized for E. coli that originates from Vibrio cholerae ’s chitin utilization program. This promoter has two ChiS binding sites and a SlmA binding site for activation– SlmA originating from both E. coli and V. cholerae’s genome. We coupled Pchb with sfGFP to report activation induced by chitin.
ChiS is a hybrid sensor kinase originating from the Vibrio cholerae chitin utilization program that is codon-optimised for E. coli . ChiS differs from prototypical two-component signal transduction pathways in that it plays the role of both the sensor kinase and the response regulator. In the presence of chitin (chitin-CBP complex bound to ChiS), ChiS favors dephosphorylation of its receiver domain, allowing it to directly bind and activate Pchb.
ChiP is a chitoporin from Vibrio cholerae that is codon-optimised for E. coli . This protein is found in the outer membrane and allows chitin oligosaccharides that were degraded by chitinases in the extracellular space to enter the periplasm.
Chitin Binding Protein (CBP)
CBP is a protein from Vibrio cholerae that is codon-optimised for E. coli . CBP is found in the periplasm where it can bind to chitin oligosaccharides and the periplasmic region of ChiS for activation. In the absence of chitin oligosaccharides, CBP binds to and inactivates ChiS by favoring phosphorylation of its receiver domain.
sfGFP (super-folded Green Fluorescent Protein) is a reporter protein that fluoresces green. We are regulating the expression of sfGFP by inducible promoter Pchb that is activated in the presence of chitin. This way, the live cell biosensor fluoresces green if chitin is present.
ChiSPY (Chitin-Sensing Pathway) Biosensor Device
The ChiSPY biosensor is a multi-transcriptional unit device that expresses the proteins expressed in V. cholerae’s one-component chitin-utilisation program coupled with a GFP reporter. This biosensor will express chitporins (ChiP) to allow chitin oligos to enter the periplasm where they then can bind to chitin-binding proteins (CBP). This chitin-CBP complex can then bind to and activate the one-component hybrid sensor kinase ChiS embedded in the inner membrane. Activated ChiS directly binds to the Pchb promoter, allowing activation of sfGFP production. This device is used as a live-cell biosensor that senses chitin oligos and produces sfGFP to signify the presence of the Bd fungus.
To measure cell burden of the individual L1 transcription units, growth curves were constructed. As can be seen from the growth curves none of the L1 transcriptional units overly burdened cell growth– all transformed cells survived. It should be noted; however, that the OD600 readings of all cultures were not standardized prior to inoculation, seen by the varying y-intercepts of all growth curves. Because of this, this data should not be used as quantitative measurements of growth kinetics, but rather a qualitative demonstration of relative growth.
The ChiS system can potentially contribute to future iGEM teams’ projects by serving as a universal noncanonical membrane-embedded one-component system. This system can act as a sensing pathway of a specific molecule to turn on transcription to produce a desired output.
The ChiS system is a noncanonical membrane-embedded one-component system that can both sense chitin oligosaccharide and directly regulate gene expression through cryptic DNA binding from the membrane. The V. cholerae that is codon-optimized for E. coli senses chitin via the hybrid sensor kinase, ChiS, to activate the output expression of the system. The hybrid sensor kinase system has many domains such as histidine and receiving phosphotransfer domain. It’s composed of a membrane-embedded histidine kinase (HK) and response regulator (RR) . The RR is a cytoplasmic partner protein within the ChiS system. Once an environmental stimulus is detected, the HK autophosphorylates histidine. The phosphate is moved to another amino acid called asaparate that is on the receiver domain of the cognate response regulator (RR) . The ChiS RR DNA binding is strengthened after dephosphorylation has occurred. Thus, the initiation of a phosphorylation cascade allows the system to signal to the cognate response receptors which allow ChiS to bring and activate Pchb for transcription.
The ChiSPY project focuses on developing a microbial biosensor by engineering E. coli to express the chitin-activated V. cholera one-component system. The system will include Pchb, ChiS, ChiP, CBP, and GFP. Pchb starts transcription to produce a GFP output. sfGFP will be activated upon the presence of chitin. Factors such as stability and toxicity helped us determine that GFP would be the optimal biomarker for our component system.
ChiP serves as a transmembrane protein transporter for the chitin oligosaccharide from the environment into the periplasm. Chitin is a component within the cell walls of empty zoosporangia that will be our biomarker. It binds to CBP which inhibits it from deactivating ChiS. The CBP serves as a phosphorylation inhibitor of ChiS that doesn’t allow ChiS to bind to Pchb to process transcription in the absence of the chitin oligosaccharide. ChiS directly binds to the Pchb which allows transcription to activate and produce GFP. To test the basal level of GFP output in the presence of Chitin, a total of 8 constructs were made to obtain data of GFP output concentration. In terms of the components of the ChiSPY construct, each DNA sequence (Pchb, CBP, ChiS, ChiP, GFP) plays a vital role in the biosensor gene circuit. More information on these constructs are discussed in our “Engineering Success” page.
 Klancher, Catherine A., et al. “ChiS Is a Noncanonical DNA-Binding Hybrid SENSOR Kinase That DIRECTLY Regulates the CHITIN Utilization Program in Vibrio Cholerae.” PNAS, National Academy of Sciences, 18 Aug. 2020, www.pnas.org/content/117/33/20180.