Difference between revisions of "Team:NEFU China/Lock Key"
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<h1 style="font-size: 65px;color: orange!important;height: 84px;">Lock & Key</h1> | <h1 style="font-size: 65px;color: orange!important;height: 84px;">Lock & Key</h1> | ||
<p> | <p> | ||
| − | Yeast: a-type yeast.<br> | + | <strong><span style="color:red" >Yeast:</span>a-type yeast.</strong><br> |
| − | Function: In order to verify the interaction between the lock (stem loop) and the key (small RNA), the EGFP or Gluc expression was determined with or without coexpression of the vector expressing the key.<br> | + | <span style="color:red">Function:</span>In order to verify the interaction between the lock (stem loop) and the key (small RNA), the EGFP or Gluc expression was determined with or without coexpression of the vector expressing the key.<br> |
| − | Vector construction:<br> | + | <span style="color:red">Vector construction:</span><br> |
| − | Gene fragments of EGFP or Gluc with the stem loop and the URA screening marker genes were inserted between HA1 and HA2 to construct the pesc-ura-Backbone-Stemloop-Gluc(or EGFP)-URA plasmid. HA1 and HA2 act as homology arms to facilitate homologous recombination, which allow the integration of the DNA with the stem loop lock sequence into the yeast genome. By verifying Gluc expression, we can prove that the keys and corresponding locks can specifically and functionally interact. | + | Gene fragments of <span style="color:orange">EGFP or Gluc</span> with the stem loop and the <span style="color:orange">URA </span>screening marker genes were inserted between HA1 and HA2 to construct the <span style="color:orange">pesc-ura-Backbone-Stemloop-Gluc(or EGFP)-URA </span>plasmid. HA1 and HA2 act as homology arms to facilitate homologous recombination, which allow the integration of the DNA with the stem loop lock sequence into the yeast genome. By verifying Gluc expression, we can prove that the keys and corresponding locks can specifically and functionally interact.<br> |
| − | Functional verification:<br> | + | <span style="color:red">Functional verification:</span><br> |
| − | 1.pesc-ura-Backbone-Stemloop-Gluc-URA plasmids were transformed into yeast. Meanwhile, plasmids with Gluc fragments and plasmids with keys were also transformed into the same yeast. Then, we measured Gluc expression using the GloMax 20/20 Luminometer to evaluate whether a lock and its corresponding key have functional interaction. In another word, we wanted to determine whether the key could specifically resolve the stem loop structure of a lock to enhance Gluc expression. Our data is presented in Figure 3A, showing that the key and the lock indeed interacted.<br> | + | <strong>1.</strong>pesc-ura-Backbone-Stemloop-Gluc-URA plasmids were transformed into yeast. Meanwhile, plasmids with Gluc fragments and plasmids with keys were also transformed into the same yeast. Then, we measured Gluc expression using the GloMax 20/20 Luminometer to evaluate whether a lock and its corresponding key have functional interaction. In another word, we wanted to determine whether the key could specifically resolve the stem loop structure of a lock to enhance Gluc expression. Our data is presented in Figure 3A, showing that the key and the lock indeed interacted.<br><br> |
<img src="https://static.igem.org/mediawiki/2018/5/50/T--NEFU_China--result12.png" style="width:60%;"><br> | <img src="https://static.igem.org/mediawiki/2018/5/50/T--NEFU_China--result12.png" style="width:60%;"><br> | ||
Figure 5. Gluc activity of pCYC-Stemloop-Gluc vector with or without coexpressed key vector. <br> | Figure 5. Gluc activity of pCYC-Stemloop-Gluc vector with or without coexpressed key vector. <br> | ||
| − | 2.pesc-ura-Backbone-Stemloop-EGFP-URA plasmids were transformed into yeast. Meanwhile, plasmids with EGFP fragments and plasmids with keys were transformed into the same yeast. The relative fluorescence intensity suggested that the key and the lock had specific interaction as shown in Figure 4B and Figure 6.<br> | + | <strong>2.</strong>pesc-ura-Backbone-Stemloop-EGFP-URA plasmids were transformed into yeast. Meanwhile, plasmids with EGFP fragments and plasmids with keys were transformed into the same yeast. The relative fluorescence intensity suggested that the key and the lock had specific interaction as shown in Figure 4B and Figure 6.<br><br> |
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| − | Figure 4. Plasmids with stem loop (B).<br></p> | + | Figure 4. Plasmids with stem loop (B).<br><br></p> |
<table id="table2"> | <table id="table2"> | ||
<tr> | <tr> | ||
Revision as of 02:24, 17 October 2018
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Lock & Key
Yeast:a-type yeast.
Function:In order to verify the interaction between the lock (stem loop) and the key (small RNA), the EGFP or Gluc expression was determined with or without coexpression of the vector expressing the key.
Vector construction:
Gene fragments of EGFP or Gluc with the stem loop and the URA screening marker genes were inserted between HA1 and HA2 to construct the pesc-ura-Backbone-Stemloop-Gluc(or EGFP)-URA plasmid. HA1 and HA2 act as homology arms to facilitate homologous recombination, which allow the integration of the DNA with the stem loop lock sequence into the yeast genome. By verifying Gluc expression, we can prove that the keys and corresponding locks can specifically and functionally interact.
Functional verification:
1.pesc-ura-Backbone-Stemloop-Gluc-URA plasmids were transformed into yeast. Meanwhile, plasmids with Gluc fragments and plasmids with keys were also transformed into the same yeast. Then, we measured Gluc expression using the GloMax 20/20 Luminometer to evaluate whether a lock and its corresponding key have functional interaction. In another word, we wanted to determine whether the key could specifically resolve the stem loop structure of a lock to enhance Gluc expression. Our data is presented in Figure 3A, showing that the key and the lock indeed interacted.
Figure 5. Gluc activity of pCYC-Stemloop-Gluc vector with or without coexpressed key vector.
2.pesc-ura-Backbone-Stemloop-EGFP-URA plasmids were transformed into yeast. Meanwhile, plasmids with EGFP fragments and plasmids with keys were transformed into the same yeast. The relative fluorescence intensity suggested that the key and the lock had specific interaction as shown in Figure 4B and Figure 6.
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Figure 4. Plasmids with stem loop (B).
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Figure 6. Plasmids with EGFP fragments and plasmids with keys were co-transformed into the yeast.
Reference
[1] Pu, Jinyue and Zinkus-Boltz, Julia and Dickinson, Bryan C. (2017) Evolution of a split RNA polymerase as a versatile biosensor platform. Nat Chem Biol 13 , 432-438
[2] Pu, Jinyue and Zinkus-Boltz, Julia and Dickinson, Bryan C. (2017) Evolution of a split RNA polymerase as a versatile biosensor platform. Nat Chem Biol 13 , 432-438
[3] Pu, Jinyue and Zinkus-Boltz, Julia and Dickinson, Bryan C. (2017) Evolution of a split RNA polymerase as a versatile biosensor platform. Nat Chem Biol 13 , 432-438
[4] Pu, Jinyue and Zinkus-Boltz, Julia and Dickinson, Bryan C. (2017) Evolution of a split RNA polymerase as a versatile biosensor platform. Nat Chem Biol 13 , 432-438
[5] Pu, Jinyue and Zinkus-Boltz, Julia and Dickinson, Bryan C. (2017) Evolution of a split RNA polymerase as a versatile biosensor platform. Nat Chem Biol 13 , 432-438
[6] Pu, Jinyue and Zinkus-Boltz, Julia and Dickinson, Bryan C. (2017) Evolution of a split RNA polymerase as a versatile biosensor platform. Nat Chem Biol 13 , 432-438 s
