Team:Hong Kong HKUST/pneumosensor/future work

Our team designed but was unable to test this module due to the limitation of not being able to work directly with Streptococcus Pneumoniae (Biosafety level 2) in our lab. This module proposes to kill Streptococcus Pneumoniae upon detection when coupled with the detection and regulation modules by releasing specific bacteriophage lytic enzymes, Cpl-1 and Pal.

Amidase (PAL)- cleaves the peptidoglycan between N-acetylmuramic acid and L-alanine

Lysozyme (CPl-1)- cleaves the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine

The enzymes are tagged with osmY (Washington 2012) via a linker to be exported out of Escherichia coli. Both enzymes have very different N-terminal catalytic sites and share a similar C-terminal cell wall attachment site, which binds to choline in both cases. Cleavage with either of these enzymes results in a weakening in the cell wall, which leads to the externalization of the cytoplasmic membrane and ultimate lysis of S. pneumoniae.

1. Put inducible promoter upstream of RBS (BBa_B0034), comX gene, and terminator (BBa_B0015). An example of inducible promoter is BBa_I0500. Hence, by putting an inducible promoter, we can tune the level of comX protein expression and characterize combox promoters (PcelA and Phelicase) on different level of comX concentration.

2. Further characterization of combox promoters (PcelA and Phelicase) by making a 3-D Graph for time, comX concentration, and Fluorescence expression.

3. Characterizing combox promoters (PcelA and Phelicase) specificity by introducing other proteins with similar structure as comX protein.

4. Continue comW construction, ligate with terminator (BBa_B0015), and introduce it to E.coli DH10B strain.

5. Characterization of comW by measuring the amount of comX protein with and without comW protein. (The function of comW protein is to protect comX protein from being degraded by ClpXP degradation enzyme)