Team:HKUST/Part3

From 2009.igem.org

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<p>Attractant production and reporter Construct </p>
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<p>Attractant Production and Reporter Construct </p>
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The attractant production pathway contains three parts, which are FUS1 promoter, ARO9 gene and FUS1 terminator. These three genes are first derived through PCR from yeast genome and then cloned into PRS426 yeast expression vector for further functional assay. <br>
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The attractant production pathway consists of three parts: FUS1 promoter, ARO9 gene and FUS1 terminator. These three genes are first derived through PCR from the yeast genome and then cloned into the PRS426 yeast expression vector for further functional assay. <br><br>
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The reporter construct also contains three parts, which are FUS1 promoter, EGFP gene and FUS1 terminator. These three genes are first derived through PCR and then cloned into PRS426 yeast expression vector for further functional assay. </p>
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The reporter construct also contains three parts: FUS1 promoter, EGFP gene and FUS1 terminator. These three genes are first derived through PCR and then cloned into the PRS426 yeast expression vector for further functional assay. <br><br></p>
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<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure05.jpg" width=518; height=272 /></a><br>
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<img src="https://static.igem.org/mediawiki/2009/c/c2/3Figure05.jpg" width=518; height=272 /></a><br><br>
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Figure 5. Attractant production pathway and Reporter detailed design</p>
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<p> I. Primer Design</p>
<p> I. Primer Design</p>
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We have designed several sets of primers for our construction, which are listed in Table 1. These mainly consist of primers for FUS1 promoter amplification, EGFP reporter amplification, ARO9 gene amplification and FUS1 terminator amplification. Primer statistics are calculated using NetPrimer. </p>
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We have designed several sets of primers for our construction, which are listed in Table 4. These primers are mainly designed for amplification of the FUS1 promoter, EGFP reporter, ARO9 gene and FUS1 terminator. Primer statistics are calculated using NetPrimer. </p><br><br>
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<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure06.jpg " width=584; height=540 /></a><br>
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Table 4 Primer sequences designed for the constructions<br>
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*Note: Restriction sites are highlighted in yellow. </p>
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<img src="https://static.igem.org/mediawiki/2009/9/9e/3Figure06.jpg" width=584; height=540 /></a><br><br>
<p> II. PCR</p>
<p> II. PCR</p>
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Three out of four genes, FUS1 promoter, FUS1 terminator and ARO9 gene are directly amplified out from yeast genomic DNA (strain YPH501). The other gene, EGFP gene, is amplified from plasmid YTK-6/Tpi/EGFP. <br>
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Three of the four desired genes, FUS1 promoter, FUS1 terminator and ARO9 gene are directly amplified from yeast genomic DNA (strain YPH501). The other gene, EGFP gene, is amplified from the plasmid YTK-6/Tpi/EGFP. <br><br>
     For optimized PCR efficiency and accuracy, Taq polymerase is chosen for amplification PCR reactions. Gradient PCR is carried out for each step to optimize the reaction. </p>
     For optimized PCR efficiency and accuracy, Taq polymerase is chosen for amplification PCR reactions. Gradient PCR is carried out for each step to optimize the reaction. </p>
<p> III. Cloning</p>
<p> III. Cloning</p>
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After the all the fragments are successfully derived through PCR, standard cloning procedures are followed to construct an integrated expression vector. <br>
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After all the genes are successfully derived through PCR, standard cloning procedures are followed to construct an integrated expression vector. <br><br>
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The PRS426 yeast expression vector is used in the expression of our construction. The FUS1 promoter is cloned into the multicloning site via Sac I and Not I digest. The ARO9 gene and the EGFP gene are cloned via blunt-end Sma I digest. FUS1 terminator is cloned via Hind III and Xho I digest. </p>
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The PRS426 yeast expression vector is used in our construction. The FUS1 promoter is cloned into the multiple cloning site by Sac I and Not I digest. The ARO9 gene and the EGFP gene are cloned by blunt-end Sma I digest. FUS1 terminator is cloned by Hind III and Xho I digest. </p>
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<br>
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<br><br><br>
<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li>
<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li>
<li><a href="https://2009.igem.org/Team:HKUST/Group3">Experimental Design</a></li>
<li><a href="https://2009.igem.org/Team:HKUST/Group3">Experimental Design</a></li>

Latest revision as of 03:46, 22 October 2009

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a

Attractant Production and Reporter Construct

The attractant production pathway consists of three parts: FUS1 promoter, ARO9 gene and FUS1 terminator. These three genes are first derived through PCR from the yeast genome and then cloned into the PRS426 yeast expression vector for further functional assay.

The reporter construct also contains three parts: FUS1 promoter, EGFP gene and FUS1 terminator. These three genes are first derived through PCR and then cloned into the PRS426 yeast expression vector for further functional assay.



I. Primer Design

We have designed several sets of primers for our construction, which are listed in Table 4. These primers are mainly designed for amplification of the FUS1 promoter, EGFP reporter, ARO9 gene and FUS1 terminator. Primer statistics are calculated using NetPrimer.





II. PCR

Three of the four desired genes, FUS1 promoter, FUS1 terminator and ARO9 gene are directly amplified from yeast genomic DNA (strain YPH501). The other gene, EGFP gene, is amplified from the plasmid YTK-6/Tpi/EGFP.

For optimized PCR efficiency and accuracy, Taq polymerase is chosen for amplification PCR reactions. Gradient PCR is carried out for each step to optimize the reaction.

III. Cloning

After all the genes are successfully derived through PCR, standard cloning procedures are followed to construct an integrated expression vector.

The PRS426 yeast expression vector is used in our construction. The FUS1 promoter is cloned into the multiple cloning site by Sac I and Not I digest. The ARO9 gene and the EGFP gene are cloned by blunt-end Sma I digest. FUS1 terminator is cloned by Hind III and Xho I digest.




  • Background
  • Experimental Design
  • Parts Design
  • Experimental Results
  • Future Work
  • References
  • HKUST