Difference between revisions of "Part:BBa K2688010:Design"

 
 
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It is possible, though not verified experimentally, that some level of ribosome readthrough occurs, and it cannot be excluded that a fusion protein "tir fragment-downstream gene" may be produced. This would explain the differing level of expression between in-frame-with-tir and out-of-frame downstream gene. If you need to avoid this possibility, use the other BioBricks LEE5 variants without tir fragment.
 
It is possible, though not verified experimentally, that some level of ribosome readthrough occurs, and it cannot be excluded that a fusion protein "tir fragment-downstream gene" may be produced. This would explain the differing level of expression between in-frame-with-tir and out-of-frame downstream gene. If you need to avoid this possibility, use the other BioBricks LEE5 variants without tir fragment.
  
−
The -35, -10 and start of translation are not well defined for this promoter sequence. We include spacer bases at the end that ensure correct translation.
+
The -35, -10 and start of translation are not well defined for this promoter sequence. We include spacer bases at the end that ensure correct translation. This sequence has a RBS included.
  
 
Be aware that due to the mechanism of repression by H-NS, which self-recruit and covers large tract of DNA, there may be interference with surrounding genes. Large spacer are recommended, or opposite strand placement of genes.
 
Be aware that due to the mechanism of repression by H-NS, which self-recruit and covers large tract of DNA, there may be interference with surrounding genes. Large spacer are recommended, or opposite strand placement of genes.

Latest revision as of 11:31, 23 September 2018


LEE5_native


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 52


Design Notes

This is as close to the native sequence as the RFC10 permits (Two illegal sites were corrected). It contains the beginning of an ORF coding for the protein tir (translocated intimin receptor - 186 of 1500 bp) starting at "ATGCCTATTGGTA". While translation of downstream gene will occurs regardless of frame, there is higher expression level when ensuring the downstream gene CDS is in frame with the tir fragment.

It is possible, though not verified experimentally, that some level of ribosome readthrough occurs, and it cannot be excluded that a fusion protein "tir fragment-downstream gene" may be produced. This would explain the differing level of expression between in-frame-with-tir and out-of-frame downstream gene. If you need to avoid this possibility, use the other BioBricks LEE5 variants without tir fragment.

The -35, -10 and start of translation are not well defined for this promoter sequence. We include spacer bases at the end that ensure correct translation. This sequence has a RBS included.

Be aware that due to the mechanism of repression by H-NS, which self-recruit and covers large tract of DNA, there may be interference with surrounding genes. Large spacer are recommended, or opposite strand placement of genes.


Source

LEE5 is a topic of research at the host lab, which provided us with the source plasmid pKK-LEE5-gfp that was then cloned to BioBrick RFC10 standard.

References