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

(→‎Design Notes)
(→‎Design Notes)
Line 14: Line 14:
 
<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 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>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, 10-1, 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>
 +
<h4>Results</h4>
  
 
===Source===
 
===Source===

Revision as of 22:52, 16 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

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.

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

Source

genomic DNA of ADH1 from S. cerevisiae

References