Part:BBa_K4815018

pDualPL1-pDual mcherry-yeGFP Low 1

Description

The functional promoter sequence (about 223 bp) generated by our AI model Pymaker is inserted into the dual-fluorescence reporter system for characterization and we describe it as pDualPL1. Random DNA yields diverse expression levels in a yeast promoter library, while we can use the episomal dual reporter system expressing a constitutive red fluorescent protein (RFP) and a variable yellow fluorescent protein (YFP) to quantify promoter activity. By adpoting the log(YFP/RFP) measured using flow cytometry, we can detect the expression rate of our synthesized promoters. The advantage of this system is its ability to eliminate the influence of plasmid copy number and the growth status of the bacterial host, thereby providing a more direct measurement of the relative expression strength of the synthesized promoters by the Pymaker.

Loci

pDualPL1 consists two parts: the synthesized core promoter and the pDual reporter scaffold. The synthesized core promoter is an 80 bp sequence generated by the Pymaker and is seated at approximately -170 to -90 upstream to the codon (which is the presumed transcription start site-TSS and is where most transcription factors binding sites lie). The pDual reporter scaffold can link the core promoter with the codon and provide restriction sites of BamH I and Xho I which make it possible for the plasmids with the scaffold to be inserted by various core promoter sequences at ease.

Usage and Biology

We used a synthetic promoter to drive the expression of the YeGFP gene on the same plasmid, while the TEF1 promoter was used in the reverse orientation to drive the expression of the mCherry gene. Additionally, we incorporated a lactose-inducible switch to enhance safety. We utilized flow cytometry to monitor the two fluorescence signals excited by different light channels and analyzed the corresponding data. We plotted the natural logarithm of the ratio of GFP to mCherry (ln(GFP/mCherry)) as a frequency distribution graph to showcase the relative expression strength of different promoters in yeast.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 396
  Illegal XbaI site found at 3641
  Illegal PstI site found at 964
  Illegal PstI site found at 2196
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 396
  Illegal NheI site found at 3407
  Illegal PstI site found at 964
  Illegal PstI site found at 2196
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 396
  Illegal BglII site found at 1927
  Illegal BamHI site found at 3604
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 396
  Illegal XbaI site found at 3641
  Illegal PstI site found at 964
  Illegal PstI site found at 2196
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 396
  Illegal XbaI site found at 3641
  Illegal PstI site found at 964
  Illegal PstI site found at 2196
  Illegal NgoMIV site found at 1826
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2886
  Illegal BsaI.rc site found at 4281
  Illegal SapI site found at 3489
  Illegal SapI site found at 4772
  Illegal SapI site found at 5372
  Illegal SapI.rc site found at 2733
  

Composition

We link PYPL1 to the plasmid framework by double enzyme digestion assay and ligation assay, the figure bellow shows the success in the assembly process. In the process pYHP1 is extracted from pDualPL1-pDual mcherry-yeGFP Higl1 and be inserted into pDualPl1-LTB-eGFP.