Difference between revisions of "Part:BBa J63005"
 
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<h2>Tianjin 2021's characterization</h2>
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<h2>Tianjin's 2021 characterization</h2>
 
<b><h3>Characterization of ADH1 Promoter’s GFP expression in SY14 yeast</h3></b>
 
<b><h3>Characterization of ADH1 Promoter’s GFP expression in SY14 yeast</h3></b>
 
<p>Group: Tianjin 2021<br>
 
<p>Group: Tianjin 2021<br>
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Author: Ruiqi Liu<br>
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Author: Ruiqi Liu, Lingwei Ding<br>
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Summary: we characterized ADH1 Promoter’s GFP expression in SY14 yeast.</p>
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Summary:we characterized ADH1 Promoter’s strength in 4742 yeast.</p>
  
 
<i><h3>Background</h3></i>
 
<i><h3>Background</h3></i>
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In order to verify whether the ADH1 promoter can express the GFP signal intensity required in experiment, we characterized the ADH1 promoter’s efficiency. We constructed a GFP fragment that was controlled by the ADH1 promoter and bound it to the chromosome genome of SY14 yeast. Firstly, we used glucose as the carbon source for enrichment, then used galactose as the carbon source for induction, and recorded the time of induction as zero point of time. The GFP expression levels of yeast at 15h, 20h,25h and 30h were measured by microplate analyzer. Meanwhile, we used SY14 yeast as control group.
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SY14 is a special kind of Artificially modified yeasts which contains only one chromosome fused by 16 chromosomes. SY14 yeast differs from normal yeast in many properties: growth rate, metabolic activity etc. We use GFP as reporter gene to test the ADH1 promoter’s strength in SY14. We constructed a GFP fragment that was controlled by the ADH1 promoter and bound it to the chromosome genome of SY14 yeast. We measured the GFP expression levels of SY14 yeast at 15h, 20h,25h and 30h by using microplate analyzer. Meanwhile, we used SY14 yeast (no GFP fragment) as control group.
  
 
<i><h3>Result</h3></i>
 
<i><h3>Result</h3></i>
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<p>We tested the expression intensity of GFP by ADH1 promoter and expressed it by fluorescence intensity /OD600. The excitation/emission wavelength of GFP in the microplate detector was set as 488/535nm. We could see clearly that ADH1 promoter is not strong enough to produce a good deal of GFP in the SY14 yeasts.</p>
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<p>We use GFP as reporter gene to test the intensity of ADH1 promoter, and draw a bar chart with fluorescence intensity /OD as the ordinate and time as the abscissa.The excitation/emission wavelength of GFP in the microplate detector was set as 488/535nm. The strength of ADH1 promoter reaches its peak at 15h. </p>
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[[File:T--Tianjin--Parts4.png|500px|thumb|left|Figure 1.Result]]
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[[File:T--Tianjin--Parts4.png|600px|thumb|left|Figure 1.Characterization of ADH1 promoter’s strength]]

Latest revision as of 05:39, 21 October 2021


yeast ADH1 promoter

ADH1 promoter from S. cerevisiae

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 NgoMIV site found at 180
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 830


Wroclaw’s 2019 characterization

Comparison of the ADH1 and TEF promoters activity

Background

Figure 1. Drop test analysis of S. cerevisiae EBY.VW4000 transformed with pRS426 plasmid containing YALI0C06424g, YALI08943g and YALI0F19184g hexose transporters from Y. lipolytica under the control of ADH1 (pA) or TEF (pT) promoter.

In order to analyze the activity of ADH1 and TEF promoters[1] and their usefulness to express sugar transporters from Yarrowia lipolytica, S. cerevisiae EBY.VW4000 (strain deleted for all hexose transporters, kindly provided by Prof. Eckhard Boles [2]) was used as a host organism. The genes encoding sugar transporters (YALI0C06424g, YALI0C08943g, YALI0F19184g) were cloned into two types of pRS426 plasmid: with S. cerevisiae ADH1 promoter or TEF promoter. The replicative plasmids were cloned into S. cerevisiae.

The transformants were cultivated in YNB medium with maltose as carbon source. After 48 h of culture, cells were centrifuged, washed twice with distilled water and its OD600 was standardized to 10. Several decimal dilutions were prepared: 100, 10-1, 10-2, 10-3, 10-4, 10-5. After the dilutions, cells were spoted in drop test on YNB agar plated with 2% glucose.

Results

The analysis of promoter strength and its usefulness for expression of hexose transporters from Y. lipolytica in S. cerevisiae EBY.VW4000 was analyzed. The 2µ plasmid (pRS426) was used with ADH1 or TEF promoter. After incubation of S. cerevisiae transformants for 48 h at 30°C growth of transformants expressing each of the three transporters under TEF promoter was observed. In turn, expression of only YALI0C06424g transporter was observed when ADH1 promoter was used. The results clearly indicated that AHD1 promoter is not strong enough and thus not appropriate for the analysis of heterologous hexose transporters in the S. cerevisiae EBY.VW4000.

Source

genomic DNA of ADH1 from S. cerevisiae

References

Wroclaw's 2019 references

[1] Partow, S., Siewers, V., Bjørn, S., Nielsen, J., & Maury, J. (2010). Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast, 27(11), 955–964. doi:10.1002/yea.1806

[2] Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg, C. P., & Boles, E. (1999). Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Letters, 464(3), 123–128. https://doi.org/10.1016/S0014-5793(99)01698-1



Tianjin's 2021 characterization

Characterization of ADH1 Promoter’s GFP expression in SY14 yeast

Group: Tianjin 2021
Author: Ruiqi Liu, Lingwei Ding
Summary:we characterized ADH1 Promoter’s strength in 4742 yeast.

Background

SY14 is a special kind of Artificially modified yeasts which contains only one chromosome fused by 16 chromosomes. SY14 yeast differs from normal yeast in many properties: growth rate, metabolic activity etc. We use GFP as reporter gene to test the ADH1 promoter’s strength in SY14. We constructed a GFP fragment that was controlled by the ADH1 promoter and bound it to the chromosome genome of SY14 yeast. We measured the GFP expression levels of SY14 yeast at 15h, 20h,25h and 30h by using microplate analyzer. Meanwhile, we used SY14 yeast (no GFP fragment) as control group.

Result

We use GFP as reporter gene to test the intensity of ADH1 promoter, and draw a bar chart with fluorescence intensity /OD as the ordinate and time as the abscissa.The excitation/emission wavelength of GFP in the microplate detector was set as 488/535nm. The strength of ADH1 promoter reaches its peak at 15h.

Figure 1.Characterization of ADH1 promoter’s strength