Difference between revisions of "Team:DTU-Denmark/Results-NAT-1"

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<div class="headlinecontainer"><h1>Results</h1></div>
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<div class="headlinecontainer"  style="font-size:5vw;"><h1><i>nat1</i> Expression</h1></div>
 
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One of the problems we encountered working with <i>Aspergillus oryzae</i>, was a lack of appropriate antifungals for selection. Consequently, we looked towards the lab hosting us for a possible solution beyond the utilization of an auxotrophic strain. One possible option for selection of <i>A. oryzae</i> is the antibiotic nourseothricin (NTC) in combination with the resistance gene <i>nat1</i> encoding the nourseothricin N-acetyl transferase (nat1) (1). Jakob Rendsvig, a Ph.D. student at DTU, kindly provided us with the plasmid, pDIV079, containing the resistance gene in a codon-optimized format under control of the <i>tef1</i> promoter. Thus, we decided to test it.
  
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<ul><li>Successfully transformed <i>A. oryzae</i> with <i>pDIVO079</i></li>
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<li>Demonstrated <i>nat1</i> as a selectable marker for <i>A. oryzae</i></li>
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<h2 class="media-heading" style="text-align: left;margin-bottom: 35px; color:#50C8E8;">Characterization</h2>
  
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<i>A. oryzae</i> RIB40 was transformed with the pDIV079 (the plasmid map can be found in fig. 1) and afterwards plated on NTC-containing transformation media at different concentrations (100 µg/ml, 200 µg/ml, 400 µg/ml, 800 µg/ml).
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<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/7/78/T--DTU-Denmark--results_nat1_pDIV079.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 1: </b> Plasmid map of pDIV079 for expression of <i>nat1</i>, kindly provided by Jakob Rendsvig. </p></figcaption>
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After 3 days of growth each concentration was replica plated onto plates containing the same concentrations as they originally were plated on. These plates can be seen below showing signs of selection (contrast with control), see fig. 2.
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<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/b/b6/T--DTU-Denmark--results_nat1_replicaplates.png
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" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 2: </b> Pictures of replica plates, from left to right: control containing no NTC, 100 µg/ml, 200 µg/ml, 400 µg/ml, 800 µg/ml.</p></figcaption>
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The transformants and a WT <i>A. oryzae</i> RIB40 were then plated onto PDA containing 800 µg/ml NTC and allowed to grow for 3 days, see fig. 2. As can be seen, clear differences in growth rate can be observed allowing for the selection of transformants, especially the formation of satellite colonies is indicative of this.
  
  
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" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 3: </b> Wild type strain and nat1 transformants plated on 800 µg/ml NTC plates. Top row: wild type strains, bottom row: nat1 transformants. Clear differences in growth rates can be seen. Interestingly the transformants produce a lot of satellite colonies.
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
 
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
 
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<p style="color:#000; font-size:14px;">(1) Rendsvig, J. (n.d.). A. oryzae, NTC / iGEM. [email].
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<a href="https://2018.igem.org/Team:DTU-Denmark/Description">Project description</a>
 
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<a href="https://2018.igem.org/Team:DTU-Denmark/Parts">Parts overview</a>
 
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Latest revision as of 02:53, 18 October 2018

nat1 Expression

One of the problems we encountered working with Aspergillus oryzae, was a lack of appropriate antifungals for selection. Consequently, we looked towards the lab hosting us for a possible solution beyond the utilization of an auxotrophic strain. One possible option for selection of A. oryzae is the antibiotic nourseothricin (NTC) in combination with the resistance gene nat1 encoding the nourseothricin N-acetyl transferase (nat1) (1). Jakob Rendsvig, a Ph.D. student at DTU, kindly provided us with the plasmid, pDIV079, containing the resistance gene in a codon-optimized format under control of the tef1 promoter. Thus, we decided to test it.

  • Successfully transformed A. oryzae with pDIVO079
  • Demonstrated nat1 as a selectable marker for A. oryzae

Characterization

A. oryzae RIB40 was transformed with the pDIV079 (the plasmid map can be found in fig. 1) and afterwards plated on NTC-containing transformation media at different concentrations (100 µg/ml, 200 µg/ml, 400 µg/ml, 800 µg/ml).

Fig. 1: Plasmid map of pDIV079 for expression of nat1, kindly provided by Jakob Rendsvig.

After 3 days of growth each concentration was replica plated onto plates containing the same concentrations as they originally were plated on. These plates can be seen below showing signs of selection (contrast with control), see fig. 2.

Fig. 2: Pictures of replica plates, from left to right: control containing no NTC, 100 µg/ml, 200 µg/ml, 400 µg/ml, 800 µg/ml.

The transformants and a WT A. oryzae RIB40 were then plated onto PDA containing 800 µg/ml NTC and allowed to grow for 3 days, see fig. 2. As can be seen, clear differences in growth rate can be observed allowing for the selection of transformants, especially the formation of satellite colonies is indicative of this.

Fig. 3: Wild type strain and nat1 transformants plated on 800 µg/ml NTC plates. Top row: wild type strains, bottom row: nat1 transformants. Clear differences in growth rates can be seen. Interestingly the transformants produce a lot of satellite colonies.

(1) Rendsvig, J. (n.d.). A. oryzae, NTC / iGEM. [email].