Part:BBa_K4815019:Design
pDualPL2-pDual mcherry-yeGFP Low 2
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 pDualPH1. 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
pDualPH1 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
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 396
Illegal BglII site found at 1927
Illegal BamHI site found at 3604 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2886
Illegal BsaI.rc site found at 4281
Illegal SapI site found at 4772
Illegal SapI site found at 5372
Illegal SapI.rc site found at 2733
Composition
We link PYPH1 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 pDualPH1-pDual mcherry-yeGFP High1 and be inserted into pDualPH1-LTB-eGFP.
