Difference between revisions of "Part:BBa K2973009"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | In our project, we used the IS6110 gene (<partinfo>BBa_K2973009</partinfo>) 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 | + | In our project, we used the IS6110 gene (<partinfo>BBa_K2973009</partinfo>) 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 <var>10<sup>-9</sup></var>nanograms in reaction times ranging from five to 20 minutes. The primers used in the amplification reactions were <partinfo>BBa_K2973016</partinfo> (forward) and <partinfo>BBa_K2973017</partinfo> (reverse). 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 (<partinfo>BBa_K2973007</partinfo>). The expected amplified product length is 136 bp. |
Latest revision as of 15:47, 21 October 2019
IS6110 (Mycobacterium tuberculosis)
This basic part is the coding sequence of the Mycobacterium tuberculosis (MTB) IS6110 gene. IS6110 is an insertion element found exclusively within the members of the Mycobacterium tuberculosis complex (MTBC), and because of this exclusivity, it has become an important diagnostic tool in the identification of MTBC species. The restriction of IS6110 to the MTBC is hypothesized to arise from the inability of these bacteria to exchange DNA. The gene IS6110 encodes for proteins responsible for the transposition activity that occurs naturally in a wide variety of organisms. IS6110 is scattered four to 25 times throughout the MTB genome.
For these reasons, IS6110 is commonly used as a DNA biomarker for the detection of MTB.
However, for safety reasons regarding the transposase that IS6110 produced naturally, we cloned only a non-functional fragment of this part into E. coli. That is prevented because the promoter-like sequence that exists in the 5' end of the part is absent in our cloned fragment, and thus transcription/translation is unable to occur. This aims to avoid the transposition of sequences of GM strains to wild type ones outside the laboratory, ensuring the highest level of biosafety.
IS6110's complete CDS is depicted with a green line and the non-functional fragment we used for our experiments (987 bp) is depicted with an orange line:
Usage and Biology
In our project, we used the IS6110 gene (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 10-9nanograms in reaction times ranging from five to 20 minutes. The primers used in the amplification reactions were BBa_K2973016 (forward) and BBa_K2973017 (reverse). 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:
PCR reaction:
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 880
Illegal XhoI site found at 1266 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 232
Illegal NgoMIV site found at 1305 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1314
Illegal BsaI.rc site found at 1295