Part:BBa_K5206002

Palsl-sfGFP

We placed the green fluorescent protein gene BBa_I746916 (sfGFP) downstream of a bidirectional promoter BBa_K5206000(PalsI) under the control of AlsR to enable real-time fluorescent signal reporting of promoter activity. Subsequently, the plasmid pCDFDuet-1-PalsI-sfGFP was constructed to verify this Part.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1092
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 377
  

Results

（1）Plasmid construction

Using pCDF-gj-Rev and pCDF-gj-For as primers, we amplified pCDFDuet1-sfGFP as a vector using PCR technology. The PalsI promoter fragment was amplified using alsI-For and GFP-Rev, and was ligated to the pCDFDuet1-sfGFP vector using Gibson assembly.

pCDFDuet-1-Palsl-sfGFP

Electrophoretic detection of PCR

After construction, the recombinant plasmid was transferred into BL21(DE3) competent cells.

（2）Fluorescence detection

After construction, the recombinant plasmid was transferred into BL21(DE3) competent cells. However, we found that the recombinant strain BL21(DE)3/pCDFDuet-1-PalsI-sfGFP obtained showed green colouration when grown on LB solid medium without the addition of D-allose, indicating the expression of green fluorescent protein.

Plasmid transformation results

We speculate that since there is only one copy of alsR on the genome of BL21(DE)3, the protein expression level brought by this expression cassette is relatively low and is not sufficient to inhibit the expression of sfGFP on the multicopy plasmid vector pCDFDuet-1-PalsI-sfGFP.