Team:HokkaidoU Japan/Project/Backbone

From 2011.igem.org

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[[File:HokkaidoU_BsaI_Backbone.png|thumb|500px|Figure1. A backbone under constitutive promoter(pTetr). Has SlrP as a injection signal, GSK tag, Bsa I Cloning Site. Desired protein can be inserted into the cloning site.]]
[[File:HokkaidoU_BsaI_Backbone.png|thumb|500px|Figure1. A backbone under constitutive promoter(pTetr). Has SlrP as a injection signal, GSK tag, Bsa I Cloning Site. Desired protein can be inserted into the cloning site.]]
   
   
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Bsa I Cloning site has unique characteristics that enable us to clone BioBrick in between two Bsa I cutting sites arranged oposit direction and retain the properties of biobrick after insertion of DNA fragment. We put it downstream of SlrP region for construction of our backbones for T3SS characterization. Bsa I cloning site is valuable part when you need to replace particular domain part at the middle of the construct.
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Bsa I Cloning site has unique characteristics that enabled us to clone BioBrick in to two flanking Bsa I restriction sites arranged in opposite directions and still retain whole constructs BioBrick properties. Cloning site was added downstream of SlrP region for construction of our backbones for T3SS characterisation. Bsa I cloning site is invaluable part when you need to repeatedly replace particular domain part at the middle of the construct.
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Bsa I restriction enzyme has unique characteristics. The enzyme cut at site different from its recognition site. Unlike EcoR I or Pst I, Bsa I regognizes GGTCTC sequence, but cuts the sequence locating 7 bases downstream from first base recognized by Bsa I of it. Which results in a 5 prime 4 base overhang structure (Fig. 2). Which is a key property for making insertion of DNA fragment in the middle of construct possible.
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Bsa I restriction enzyme is classified as Type IIs restriction endonuclease. The unique property of this class is that recognition site is apart of restriction site . Unlike EcoR I or Pst I, Bsa I recognises GGTCTC sequence, but cuts the sequence located 7 bases downstream from first base recognised by Bsa I. Which results in a 5 prime 4 base overhang structure (Fig. 2). This is a key property that enables insertion of BioBrick in the middle of construct possible.
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Of course there are other restriction endonucleases that exhibit same properties but Bsa I. Using such enzymes, it is possible to add additional insertion sites in the same plasmid.
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Of course there are other restriction endonucleases that exhibit same properties. Using other enzymes of this class it is possible to add additional cloning sites in the same construct.
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For designing the construct Bsa I a cloning site we alocated Not I like sequence and Spe I like sequence downstream of each Bsa I site.(Fig. 3).
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Designing the construct Bsa I a cloning site we allocated Not I like overhang and Spe I like overhang downstream of each Bsa I site.(Fig. 3).
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Revision as of 12:01, 15 December 2011

Contents

  • Abstract
  • What`s T3SS
    Detailed information about T3SS and summary of our achievements on iGEM 2010
  • Injection assay using onion cells
    Experiments using plant cells are easier to perform than with mammalian ones
  • Ready-to-inject backbone and Bsa I cloning site
    Ready-to-inject backbone and Bsa I cloning site enables easy fusion of T3S signal and protein
  • GSK tag system
    A neat injection assay using GSK tag, which can specifically detect successfully injected proteins
  • Bsa I cloning site, RFC submission
    Detailed documentation of costructing a BioBrick cloning site a BioBrick!

Ready-to-inject backbone and Bsa I cloning site

Figure1. A backbone under constitutive promoter(pTetr). Has SlrP as a injection signal, GSK tag, Bsa I Cloning Site. Desired protein can be inserted into the cloning site.

Bsa I Cloning site has unique characteristics that enabled us to clone BioBrick in to two flanking Bsa I restriction sites arranged in opposite directions and still retain whole constructs BioBrick properties. Cloning site was added downstream of SlrP region for construction of our backbones for T3SS characterisation. Bsa I cloning site is invaluable part when you need to repeatedly replace particular domain part at the middle of the construct.

Bsa I restriction enzyme is classified as Type IIs restriction endonuclease. The unique property of this class is that recognition site is apart of restriction site . Unlike EcoR I or Pst I, Bsa I recognises GGTCTC sequence, but cuts the sequence located 7 bases downstream from first base recognised by Bsa I. Which results in a 5 prime 4 base overhang structure (Fig. 2). This is a key property that enables insertion of BioBrick in the middle of construct possible. 

Fig. 2 
 5'...GGTCTCN^.......3'
 3'...CCAGAGNNNNN^...5'

Of course there are other restriction endonucleases that exhibit same properties. Using other enzymes of this class it is possible to add additional cloning sites in the same construct.

Designing the construct Bsa I a cloning site we allocated Not I like overhang and Spe I like overhang downstream of each Bsa I site.(Fig. 3). 

Fig. 3
          Bsa I    Not I'           Spe I'
           -->
 5'...GG GGTCTC A^GGCC ….........^CTAG A GAGACC...3'
 3'...CC CCAGAG T CCGG^TCCGGCCGCT GATC^T CTCTGG...5' 

 5'...GG GGTCTC A                 CTAG A GAGACC...3'
 3'...CC CCAGAG T CCGG                 T CTCTGG...5'
                                          <--
                                         Bsa I

Please pay attention to remove Bsa I site from DNA sequence in BioBricks when you use this plasmid backbone.

For domain fusion, removal of stop codon existing in prefix sequence in essential. Usage of DNA primer that has a sequence for biofusion is required for amplification of inserted DNA fragment.

RFC submission

We have submitted this method as BBF RFC 87. For more details about RFC submission, please see also here.

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