Difference between revisions of "Part:BBa K2292004"

Latest revision as of 03:50, 2 November 2017

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)

The DNA size we expect to show up is 2563 bp, hence from the electrophoresis gel photos above, we can say that the plasmid DNA in well number 1, 3, 4, 8, 9, 10, 11, 13, 16, 19, 21, 24, 25, 27, 28 are those we want, and the corresponding patch are those that are successfully transformed.

Figure 3 Agarose gel electrophoresis of PCR product (pSBBS4S)

The DNA size we expect to show up is 1344 bp, hence from the electrophoresis gel photos above, we can say that the plasmid DNA in all wells, except well number 5,17, 18 ,31, are the ones we want, and the corresponding patch are those bacteria that has been successfully transformed.

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