Part:BBa_K316000

Reverse strand coding Enhanced LacI-hyperspank promoter

This part is a modified version of hyper-spank promoter for B.subtilis BBa_K143015. Hyper-spank promoter is repressed by transcriptional repressor LacI BBa_K143033 and can be induced by addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG). Constitutive expression of LacI is required for repression.

Promoter Design The position and sequence of LacI binding was designed using existing knowledge. The stochastic nature of transcriptional repressors usually leads to background transcription. In order to minimise background the binding sites and the distance between them have been optimised.

Stronger binding The natural LacI operator has 3 binding sites, all of which have variations in the binding sequences1. Perfectly symmetric binding sequence was shown to have 8-fold higher binding2 compared to wild type sequences. The aattgtgagc gctcacaatt sequence has been shown to be optimal for LacI binding3.

Optimal distance Due to the tetrameric nature of LacI it can simulataneously bind to multiple regions in the genome. Binding at multple sites can produce much stronger repression1 by increasing local LacI concentrations. Due to the helical nature of DNA the distance between the operator sites plays an important role in the strength of repression. Maximal repression at 70.5bp, second strongest at 92.5bp and third at 115.5bp3.

This is a planning part, the sequence was used to produce BBa_K316012 and BBa_K316010

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Unknown
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

<biblio>

1. 1 pmid=8045263
2. 2 pmid=6369330
3. 3 pmid=8632456

</biblio>References

For more information about our project please visit our wiki [http://2010.igem.org/Team:Imperial_College_London].