Difference between revisions of "Part:BBa J63005:Design"

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<h2>Wroclaw’s 2019 characterization</h2>
 
<h2>Wroclaw’s 2019 characterization</h2>
 
<h3>Comparison of the ADH1 and TEF promoters activity</h3>   
 
<h3>Comparison of the ADH1 and TEF promoters activity</h3>   
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<u>''Background''</u>
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<h4><i>Background</i></h4>[[Image:Wro_drop.png|400px|thumb|Figure 1. ''Drop test analysis of <i>S. cerevisiae</i> 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.  '']]
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<p>In order to analyze the activity of ADH1 and TEF promoters 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) 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.</p>
+
<p>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.</p>
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<p>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.</p>
+
<p>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<sup>-1</sup>, 10<sup>-2</sup>, 10<sup>-3</sup>, 10<sup>-4</sup>, 10<sup>-5</sup>. After the dilutions, cells were spoted in drop test on YNB agar plated with 2% glucose.</p>
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<h4>Results</h4>
+
<h4><i>Results</i></h4>
 +
<p>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.</p>
  
 
===Source===
 
===Source===
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===References===
 
===References===
 +
 +
<h5>Wroclaw's 2019 references</h5>
 +
 +
[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

Latest revision as of 20:13, 19 October 2019


yeast ADH1 promoter


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


Design Notes

This sequence corresponds to the 1457 bp directly upstream of the start codon of the ADH1 gene in S. cerevisiae.

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