Difference between revisions of "Part:BBa K2973009:Design"
 
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===Design Notes===
 
===Design Notes===
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IS6110 has normally two open reading frames. OrfA (327 bp) and orfB (927 bp) are consecutive ORFs partially overlapping in the relative translational reading frames 0 and −1, respectively. For the purposes of our project, we designed primers for orfB only.
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IS6110 has normally two open reading frames. OrfA (327 bp) and orfB (927 bp) are consecutive ORFs partially overlapping in the relative translational reading frames 0 and −1, respectively. For the purposes of our project, we designed primers for orfB only. To ensure maximum biosafety, we cloned only a non-functional fragment of the part into E. coli DH5-alpha via TA cloning. This guarantees safety for the environment for potential GM DNA transpositions, as well as enough DNA material for us to work on our project.
  
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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:
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      <img src="https://2019.igem.org/wiki/images/a/a4/T--Thessaly--Registry_IS6110.png" width="900"
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Latest revision as of 16:34, 20 October 2019


IS6110 (Mycobacterium tuberculosis)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 880
    Illegal XhoI site found at 1266
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 232
    Illegal NgoMIV site found at 1305
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1314
    Illegal BsaI.rc site found at 1295


Design Notes

IS6110 has normally two open reading frames. OrfA (327 bp) and orfB (927 bp) are consecutive ORFs partially overlapping in the relative translational reading frames 0 and −1, respectively. For the purposes of our project, we designed primers for orfB only. To ensure maximum biosafety, we cloned only a non-functional fragment of the part into E. coli DH5-alpha via TA cloning. This guarantees safety for the environment for potential GM DNA transpositions, as well as enough DNA material for us to work on our project.

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: HTML img Tag


Source

NCBI Mycobacterium tuberculosis Genome, Strain H37Rv, Accession Number: NC_000962

References

D Thierry, A Brisson-Noël, V Vincent-Lévy-Frébault, S Nguyen, J L Guesdon, B Gicquel, Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis (1990), Journal of Clinical Microbiology

Thabet, S., & Souissi, N. (2016). Transposition mechanism, molecular characterization and evolution of IS6110, the specific evolutionary marker of Mycobacterium tuberculosis complex. Molecular Biology Reports, 44(1), 25–34.doi:10.1007/s11033-016-4084-x