Difference between revisions of "Part:BBa K3221200"
 
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GAL system is a galactose-mediated induction system in yeast. To be specific, GAL1 promoter can induce downstream gene expression under the control of GAL4 protein. What’s more, in order to simplify the verification of GAL1 promoter function, we chose the yeGFP as a reporter instead of Cns1 and Cns2 in our project.  
 
GAL system is a galactose-mediated induction system in yeast. To be specific, GAL1 promoter can induce downstream gene expression under the control of GAL4 protein. What’s more, in order to simplify the verification of GAL1 promoter function, we chose the yeGFP as a reporter instead of Cns1 and Cns2 in our project.  
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Then, we designed to culture transformed yeast and WT in YPD medium and YPG medium separately. Through detecting green fluorescence to verify the function of GAL4 promoter.
 
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[[File: T--SCU-China--scu-pGAldelay2019.png|450px|]]
 
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'''Fig.1 Fluorescence observation''' ''' A1 and B1: WT in YPG；A2 and B2 : Transformed in YPD；A3, A4, B3 and B4: Transformed in YPG. Visible Fluorescence in B3 and B4. '''
 
  
 
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===Usage and Biology===
 
===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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'''Design: '''
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In order to demonstrate the function of GAL1 promoter, we cultured transformed yeast and WT in YPD medium and YPG medium separately. Through detecting green fluorescence both in Fluorescence microscope(Figure.1 (a)) and plate reader(Figure.1 (b)) to verify the function of GAL1 promoter.
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[[File:T--SCU-China--scu-result-delay expression-Figure 4(right).png|600px |]]
  
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'''Figure. 1 ''' '''The green fluorescence of S.cerevisiae BY4741 transformed pYES2-yeGFP which was induced by galactose. (a) Fluorescence observation of <i>S.cerevisiae</i> BY4741 transformed pYES2-yeGFP. ''' ''' Series 1: BY4741 WT in YPD mediuM, Series 2: BY4741 WT in YPG medium, Series 3:  BY4741 transformed BY4741 in YPD medium, Series 4: Transformed BY4741 in YPG medium. Visible Fluorescence in Series 4. ''' ''' (b) The fluorescence measurement of <i>S.cerevisiae</i>BY4741 transformed pYES2-yeGFP. '''  ''' YPD: YPD medium, YPG: YPG medium, BY4741 WT: <i>S.cerevisiae</i>, BY4741 wildtype, BY4741 transformed: <i>S.cerevisiae</i>, BY4741 transformed plasmid pYES2-yeGFP, excitation light is 488nm and emission light is 525nm，***: P<0.001. '''
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'''Result & conclusion: '''
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Generally, we verified the function of GAL1 promoter by observing green fluorescence in Figure. 1 (a) B4. The GAL1 promoter can be induced to express of downstream gene (yeGFP in this experiment) by galactose.
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===Functional Parameters===
 
===Functional Parameters===
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Latest revision as of 15:26, 21 October 2019


Galactose-mediated induction expression system for S.cerevisiae, yeGFP as a reporter

GAL system is a galactose-mediated induction system in yeast. To be specific, GAL1 promoter can induce downstream gene expression under the control of GAL4 protein. What’s more, in order to simplify the verification of GAL1 promoter function, we chose the yeGFP as a reporter instead of Cns1 and Cns2 in our project.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 150
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1199

Design:

In order to demonstrate the function of GAL1 promoter, we cultured transformed yeast and WT in YPD medium and YPG medium separately. Through detecting green fluorescence both in Fluorescence microscope(Figure.1 (a)) and plate reader(Figure.1 (b)) to verify the function of GAL1 promoter.

T--SCU-China--scu-result-delay expression-Figure 4(right).png

Figure. 1 The green fluorescence of S.cerevisiae BY4741 transformed pYES2-yeGFP which was induced by galactose. (a) Fluorescence observation of S.cerevisiae BY4741 transformed pYES2-yeGFP. Series 1: BY4741 WT in YPD mediuM, Series 2: BY4741 WT in YPG medium, Series 3: BY4741 transformed BY4741 in YPD medium, Series 4: Transformed BY4741 in YPG medium. Visible Fluorescence in Series 4. (b) The fluorescence measurement of S.cerevisiaeBY4741 transformed pYES2-yeGFP. YPD: YPD medium, YPG: YPG medium, BY4741 WT: S.cerevisiae, BY4741 wildtype, BY4741 transformed: S.cerevisiae, BY4741 transformed plasmid pYES2-yeGFP, excitation light is 488nm and emission light is 525nm，***: P<0.001.

Result & conclusion:

Generally, we verified the function of GAL1 promoter by observing green fluorescence in Figure. 1 (a) B4. The GAL1 promoter can be induced to express of downstream gene (yeGFP in this experiment) by galactose.