Difference between revisions of "Part:BBa K2923019:Experience"
 
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===Introduction===
 
===Introduction===
  
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To validate the designed constructs we decided to test two different aptazymes: Theophylline switch-off (stop auto-cleavage in presence of ligand) and Guanine switch-on (active auto-cleavage in presence of ligand). For negative control, we choose an inactive form of aptazyme: mutation at the ribozyme domain. iGEM Strasbourg designed construct requires the use of aptazyme linked to MS2 and PP7 stem-loops. The following results described MS2-MS2-Aptazyme-PP7-PP7 activity in in-vitro and in-vivo assays.
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To validate the designed constructs we decided to test Theophylline switch-off (stop auto-cleavage in presence of ligand). For negative control, we choose an inactive form of aptazyme: mutation at the ribozyme domain. iGEM Strasbourg designed construct requires the use of aptazyme linked to MS2 and PP7 stem-loops. The following results described MS2-MS2-Aptazyme-PP7-PP7 activity in in-vitro and in-vivo assays.
  
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===In-vitro assay of BBa_K2923021===
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===In-vitro assay of BBa_K2923019===
  
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In order to check MS2-MS2-Guanine-PP7-PP7 cleavage activities, in-vitro transcription products were analyzed by 2% agarose gel with urea (Figure 1).
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In order to check MS2-MS2-Theophylline-PP7-PP7 cleavage activities, in-vitro transcription products were analyzed by 2% agarose gel with urea (Figure 1).
  
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[[Image: Gua gel-1.jpg|300px|thumb|center| Figure 1: Aptazyme catalytic activity assay. Guanine aptazyme were transcribed in vitro for 1h30. RNA was analyzed in an 8% polyacrylamide gel with urea. The inactive form of Guanine aptazyme represents the whole RNA and corresponds to the band of 138 pb. Cleaved aptazyme represents the two parts of the aptazyme Guanine (28 pb + 110 pb).
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[[Image: Theo gel-2.jpg|300px|thumb|center| Figure 1: Aptazyme catalytic activity assay. Theophylline aptazyme were transcribed in-vitro for 1h30. RNA was analyzed in an 8% polyacrylamide gel with urea. The inactive form of Theophylline aptazyme represents the whole RNA and corresponds to the band of 94 pb. Cleaved aptazyme represents the two parts of the aptazyme Theophylline (18 pb + 76 pb).
 
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Inactive aptazyme did not present aptazyme catalytic activity, which confirms their use as an efficient negative control.
 
Inactive aptazyme did not present aptazyme catalytic activity, which confirms their use as an efficient negative control.
  
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To see in-vivo assays results of MS2-MS2-Guanine-PP7-PP7 aptazymes see <partinfo>BBa_K2923020</partinfo>.
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To see in-vivo assays results of MS2-MS2-Theophylline-PP7-PP7 aptazymes see <partinfo>BBa_K2923018</partinfo>.
  
  

Latest revision as of 18:20, 16 October 2019

Introduction

To validate the designed constructs we decided to test Theophylline switch-off (stop auto-cleavage in presence of ligand). For negative control, we choose an inactive form of aptazyme: mutation at the ribozyme domain. iGEM Strasbourg designed construct requires the use of aptazyme linked to MS2 and PP7 stem-loops. The following results described MS2-MS2-Aptazyme-PP7-PP7 activity in in-vitro and in-vivo assays.

In-vitro assay of BBa_K2923019

In order to check MS2-MS2-Theophylline-PP7-PP7 cleavage activities, in-vitro transcription products were analyzed by 2% agarose gel with urea (Figure 1).

Figure 1: Aptazyme catalytic activity assay. Theophylline aptazyme were transcribed in-vitro for 1h30. RNA was analyzed in an 8% polyacrylamide gel with urea. The inactive form of Theophylline aptazyme represents the whole RNA and corresponds to the band of 94 pb. Cleaved aptazyme represents the two parts of the aptazyme Theophylline (18 pb + 76 pb).

Inactive aptazyme did not present aptazyme catalytic activity, which confirms their use as an efficient negative control.

To see in-vivo assays results of MS2-MS2-Theophylline-PP7-PP7 aptazymes see BBa_K2923018.


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