Difference between revisions of "Part:BBa K2973016"
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IS6110 is an insertion element found exclusively within the members of the <i>Mycobacterium tuberculosi</i> complex (MTBC). This part is a forward primer with a T7 Promoter (BBa_J64997) on its 5' end. The primer is designed to amplify the IS6110 gene (BBa_K2973009), along with its reverse primer (BBa_K2973017).
 
IS6110 is an insertion element found exclusively within the members of the <i>Mycobacterium tuberculosi</i> complex (MTBC). This part is a forward primer with a T7 Promoter (BBa_J64997) on its 5' end. The primer is designed to amplify the IS6110 gene (BBa_K2973009), along with its reverse primer (BBa_K2973017).
  
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===Usage and Biology===
 
===Usage and Biology===
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In our project, we used the IS6110 gene ([[Part:BBa_K2973009]]) as a DNA template, in order to perform isothermal amplification reactions with Recombinase Polymerase Amplification (RPA). Using this method, we were able to detect quantities as low as 10 to the minus 9 nanograms in reaction times ranging from five to 20 minutes. This primer (BBa_K2973016) was used along with its reverse primer (BBa_K2973017) for the amplification reactions. These primers include 5' overhangs ( a T7 promoter and a trigger sequence) to allow for transcription by a T7 RNA Polymerase and translation by a toehold-switch regulated system encoding for a reporter gene (BBa_K2973007). The expected amplified product length is 136 bp.
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RPA reaction after clean up of the amplified product:
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      <title>HTML img Tag</title>
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  <body>
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      <img src="https://2019.igem.org/wiki/images/7/7e/T--Thessaly--Registry_Figure6.png" width="502"
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        height="225">
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</html>
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PCR reaction:
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<html>
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  <head>
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      <title>HTML img Tag</title>
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  </head>
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  <body>
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      <img src="https://2019.igem.org/wiki/images/c/c7/T--Thessaly--Results_Figure3A.jpg" width="285"
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        height="350">
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  </body>
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</html>
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Revision as of 16:56, 20 October 2019


IS6110 Forward Primer with 5' Overhang

IS6110 is an insertion element found exclusively within the members of the Mycobacterium tuberculosi complex (MTBC). This part is a forward primer with a T7 Promoter (BBa_J64997) on its 5' end. The primer is designed to amplify the IS6110 gene (BBa_K2973009), along with its reverse primer (BBa_K2973017).

Usage and Biology

In our project, we used the IS6110 gene (Part:BBa_K2973009) as a DNA template, in order to perform isothermal amplification reactions with Recombinase Polymerase Amplification (RPA). Using this method, we were able to detect quantities as low as 10 to the minus 9 nanograms in reaction times ranging from five to 20 minutes. This primer (BBa_K2973016) was used along with its reverse primer (BBa_K2973017) for the amplification reactions. These primers include 5' overhangs ( a T7 promoter and a trigger sequence) to allow for transcription by a T7 RNA Polymerase and translation by a toehold-switch regulated system encoding for a reporter gene (BBa_K2973007). The expected amplified product length is 136 bp.


RPA reaction after clean up of the amplified product:

HTML img Tag


PCR reaction:

HTML img Tag


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]