Difference between revisions of "Part:BBa K4815011:Experience"

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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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<partinfo>BBa_K4815019 short</partinfo>
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how you used this part and how it worked out.
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===Applications of BBa_K4815011===
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<partinfo>BBa_K4815019 SequenceAndFeatures</partinfo>
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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. The figure bellow shows success in utilizing the plasmids in detecting fluorescence intensity.
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===Design Notes===
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Our Pymaker synthesized core promoter sequence is extracted through PCR, and introducing the restriction sites ofBamH I and Xho I, the same as the scaffold. The scaffold is a preserved sequence in all PYPLs. It is a structure that we learned and utilized from previous research that 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. The scaffold is already maintained in the plasmid framework yeast_DualReporter(AddGene: 127546),using this assay we create a promoter library that can drive the high expression of YeGFP and be used quickly for the detection of expression rate.
 
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<figcaption>pA-core PYPH/PYPL-pT </figcaption>
 
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===User Reviews===
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===Source===
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<!-- DON'T DELETE --><partinfo>BBa_K4815011 StartReviews</partinfo>
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<!-- Template for a user review
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Originated from the framework yeast_DualReporter(AddGene: 127546)
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===References===
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[1] Liu, X., et al., Dual Fluorescent Protein (yEGFP/DsRed-Express-2) Bioassay System for Rapid Screening for Chemical Mutagens Based on RNR3 Regulation in Saccharomyces Cerevisiae. Biomed Environ Sci, 2021. 34(5): p. 421-424.
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<partinfo>BBa_K4815011 AddReview number</partinfo>
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Enter the review inofrmation here.
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<!-- DON'T DELETE --><partinfo>BBa_K4815011 EndReviews</partinfo>
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Revision as of 14:46, 12 October 2023


pDualPL2-pDual mcherry-yeGFP Low 2


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 4772
    Illegal SapI site found at 5372
    Illegal SapI.rc site found at 2733


Design Notes

Our Pymaker synthesized core promoter sequence is extracted through PCR, and introducing the restriction sites ofBamH I and Xho I, the same as the scaffold. The scaffold is a preserved sequence in all PYPLs. It is a structure that we learned and utilized from previous research that 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. The scaffold is already maintained in the plasmid framework yeast_DualReporter(AddGene: 127546),using this assay we create a promoter library that can drive the high expression of YeGFP and be used quickly for the detection of expression rate.

pA-core PYPH/PYPL-pT

Source

Originated from the framework yeast_DualReporter(AddGene: 127546)

References

[1] Liu, X., et al., Dual Fluorescent Protein (yEGFP/DsRed-Express-2) Bioassay System for Rapid Screening for Chemical Mutagens Based on RNR3 Regulation in Saccharomyces Cerevisiae. Biomed Environ Sci, 2021. 34(5): p. 421-424.