Part:BBa_K3487014
A fluorescent protein gene regulated by a two-component system
a detection system on CSP induction
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 79
Illegal NgoMIV site found at 145
Illegal AgeI site found at 620
Illegal AgeI site found at 986
Illegal AgeI site found at 1687 - 1000COMPATIBLE WITH RFC[1000]
2020 SZPT-CHINA
Construction and Identification of detection system based on comD and comE induction of S.mutans
The pelB-comD, comE and nlmC-sfGFP genes were cloned into pET28a(+) vector. Positive clones were selected from LB plates containing 50μg/mL kanamycin sulfate for double restriction analysis and PCR analysis. As shown in Fig.1-3, the fragment size is the same as pelB-comD, comE and nlmC-sfGFP. The results showed that the recombinant expression vector pET28a(+)-pelB-comD-comE-nlmC-sfGFP (pET28a-DEG) was successfully constructed.
The result of the detection system on CSP induction
he recombinant strain E. coli BL21 (DE3) with pET28a-DEG vector works as a detector. Different concentrations of CSP were added to the culture medium for checking the output variations. When the concentration of CSP reaches 0.5mg/ml, the comD-CSP complex would Phosphorylation of comE, phosphorylated comE and nlmC promoter combined to start the expression of the downstream fluorescent gene GFP.（Fig.4). The fluorescence intensity gradually increased as the culture time prolonged. After 24h, there was a significant difference in fluorescence intensity. This result showed that the density sensing system in E. coli detector was successfully functional.
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