Part:BBa_K2292004

CSP detection through GFP intensity

https://parts.igem.org/Part:BBa_K2292003

Our project aims to detect the risk of caries through three parameters: Competence stimulating peptide, CSP concentration, lactate level, pH value.

K2292003 is the part that we used to detect one of our target molecule, CSP. It will produce membrane receptor, comD. ComD then will bind to CSP. The comD-CSP complex will phosphorylate comE. The phophorylated comE, finally will bind to a comE responsive promoter( on this part) and produce GFP. Through GFP intensity, we will learn the concentration of CSP.

Figure 1 Mechanism of CSP detection

Figure 2 Agarose gel electrophoresis of PCR product (pSBBS1C)

Figure 3 Agarose gel electrophoresis of PCR product (pSBBS4S)

For the measurement of the fluorescent plate reader (excitation: 485 nm, emission: 520 nm), we found out that the best amount of bacteria for measuring, the CSP conc. is at the value of OD600 = 0.2 after a series of experiment.

Figure 4 Three-dimension diagram (Fluorescence-CSP concentration-Time)

The intensity of fluorescence slowly drops after a sharp rise in a small amount of time, hence we can tell that the reaction starts right away as soon as CSP is added.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 786