Team:UI-Indonesia/Project/Hunting

Sensing

CAI-1 ((S)-3-hydroxytridecan-4-one) is a quorum sensing signal molecule that is specific for V. cholerae intragenera. It is generated by CqsA and is the main regulatory autoinducer used by V. cholerae aside from the usual autoinducer-2 from the other gram negative bacteria. CqsS histidine kinase transmembrane receptor is a dominant from two quorum sensing receptor in V. cholerae, different with the other vibrio family which integrated three quorum sensing receptor. This receptor will receive CAI-1 in the DHP, CA, and C-terminal receiver domain which will induce auto phosphorylation of His194 (H1). The phosphorylation will cascade into the Asp618 (D1) at CqsS receiver domain, His58 (H2) at LuxU, Asp47 (D2) at LuxO, and inhibiting the expression of Qrr1-4 which will activates HapR (competence and protease regulator) and inhibiting AphA (biofilm regulator)^[14].

Our team would like to overexpressed the CqsS receptor in E.coli transmembrane. But overexpressed transmembrane receptor is is difficult and highly toxic for the cells. Therefore we use translocator protein to help the fusion of CqsS into transmembrane region. YaiN and YbeL are short hydrophobic bacterial proteins, they fused to the end of membrane protein to serve as fascilitating factors (translocator) purification and expression. Most protein were expressed in moderet to high level and detergent solubilization combined with short purification process and stable product^[22].

Motility

The motility of E.coli is supported by flagella. These flagella is located around the wall cell of E.coli. The E.coli’s flagella movement can be occured in two directions, clockwise and counterclockwise. If flagella rotates in clockwise direction, E.coli will tumbles, and if flagella rotates in counterclockwise direction, E.coli will runs (smooth swimming). The determinant of destination or direction of E.coli motility, we can devide it as two types of stimuli. These stimuli are positive stimuli (E.coli comes forward to attractant) and negative stimuli E.coli goes away from the attractant^[2].

E. coli’s flagella movement is influenced by some genes such as cheA, cheB, cheR, cheW, cheY, cheZ, fliG, fliM, fliN, and some receptor genes (tap, tar, tsr, trg, aer). These genes cooperates with each other to control the chemotaxis of E. coli such as to figure out the attractant as a chemoattractants (positive stimuli) or as a chemorepellents (negative stimuli), give the motility respond to go away or approach, and control the flagella direction. E. coli tumbles because of cheY gene which is phosphorylated (cheY-P; combine with P element), and this case can be stopped by cheZ gene expression which can dephosphorylated that cheY gene, so E. coli can run forward (smooth swimming)^[2]. We can make E.coli which has great motility ability (not tumbles). So, we need cheZ over expression which can dephosphorylated cheY-P. To make E.coli which can move to a specific attactant, we can add a sensitive promoter to a substance that we want. When there’s Vibrio cholerae, our E.coli will detect it by the presence of CAI-1 molecules. So, we can add promoter that sense CAI-1 molecules, and voila! Our E. coli will run forward to kill Vibrio cholerae.