Difference between revisions of "Part:BBa K316000"
 
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'''Stronger binding'''
 
'''Stronger binding'''
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The natural LacI operator has 3 binding sites, all of which have variations in the binding sequences. Perfectly symmetric binding sequence was shown to have10-fold higher binding compared to wild type sequences. The '''aattgtgagc gctcacaatt''' sequence has been shown to be optimal for LacI binding Muller 1996 Oehler 1994.
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The natural LacI operator has 3 binding sites, all of which have variations in the binding sequences<cite>1</cite>. Perfectly symmetric binding sequence was shown to have 8-fold higher binding<cite>2</cite> compared to wild type sequences. The '''aattgtgagc gctcacaatt''' sequence has been shown to be optimal for LacI binding<cite>3</cite>.
  
 
'''Optimal distance'''
 
'''Optimal distance'''
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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 repression (muller 1996) 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 115.5bp
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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 repression<cite>1</cite> 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 115.5bp<cite>3</cite>.
  
 
This is a planning part, the sequence was used to produce <bbpart>BBa_K316012</bbpart> and <bbpart>BBa_K316010</bbpart>
 
This is a planning part, the sequence was used to produce <bbpart>BBa_K316012</bbpart> and <bbpart>BBa_K316010</bbpart>
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<!-- Uncomment this to enable Functional Parameter display
 
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===Functional Parameters===
 
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<partinfo>BBa_K316000 parameters</partinfo>
 
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<!-- -->
 
  
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===References===
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<biblio>
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#1 pmid=8045263 Oehler et.al 1994 EMBO J. 15;13(14):3348-55.
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#2 pmid=6369330 Simons et.al 1984 Proc Natl Acad Sci U S A. 81(6):1624-8
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#3 pmid=8632456 Müller et.al 1996 J Mol Biol. 22;257(1):21-9.
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</biblio>References
  
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<biblio>
 
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#1 http://2010.igem.org/Team:Imperial_College_London
 
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#2 pmid=14597697
 
  
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</biblio>
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----
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For more information about this part of our project please visit our [http://2010.igem.org/Team:Imperial_College_London/Strategy wiki] or [http://2010.igem.org/Team:Imperial_College_London/Tour/Page_One take the tour] to learn more about the project.

Latest revision as of 03:21, 28 October 2010

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
    COMPATIBLE WITH RFC[10]
  • 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 Oehler et.al 1994 EMBO J. 15;13(14):3348-55.
  2. 2 pmid=6369330 Simons et.al 1984 Proc Natl Acad Sci U S A. 81(6):1624-8
  3. 3 pmid=8632456 Müller et.al 1996 J Mol Biol. 22;257(1):21-9.

</biblio>References


For more information about this part of our project please visit our [http://2010.igem.org/Team:Imperial_College_London/Strategy wiki] or [http://2010.igem.org/Team:Imperial_College_London/Tour/Page_One take the tour] to learn more about the project.