Part:BBa_K2292003

comDE two components signal transduction system

This is a composite used for producing comD, comE, two essential protein for the CSP two components signal transduction system. Originated from Streptococcus mutans. ComD is a kinase and membrane-associated receptor. After comD binded to the quorum sensing molecule CSP, the comD-CSP complex will phosphorylate comE. Then, the phosphorylated comE can turn on the transcription of downstream gene. FLAG-TAG is added after comE and c-Myc is added after comD.

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 patches 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 patches 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 268
  Illegal XhoI site found at 123
  Illegal XhoI site found at 153
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]