Team:HokkaidoU Japan/Project/Backbone

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==Ready-to-inject backbone and Bsa I cloning site==
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[[File:HokkaidoU_Japan_2011_GSK_Backbone_lv.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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[[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==
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Bsa I Cloning site is unique in a sense that you can clone BioBrick into a middle of a construct and still retain the properties of biobrick. We used it to construct our backbones for T3SS characterization. Bsa I cloning site is valuable part when you need change particular part in the middle of the construct. It was designed that inserted biobrick would be fused to preceding signals.
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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 is in distinguish group of enzyme which cutting site is different from recognition site. Unlike EcoR I or Pst I, Bsa I regognizes GGTCTC sequence but cuts the sequence 1 base further ahead of it. Which results in a 5 prime 4 base overhang(Fig). Which is the key future making insertion 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.
<pre>
<pre>
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5'...GGTCTCN^.......3'
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Fig. 2
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3'...CCAGAGNNNNN^...5'
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5'...GGTCTCN^.......3'
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3'...CCAGAGNNNNN^...5'
</pre>
</pre>
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You can manipulate the sequence of overhang as you like. By if you construct sequence GGTCTCNAATTN you can make it to ligate with EcoR I digested strand. As long as NAATTN won't become GAATTG it wouldn't not be digested by EcoR I and that’s the beauty of it.
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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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Of course there are other restriction endonucleases that exhibit same properties but Bsa I. You cannot use more than one Bsa I cloning site per construct. However, using other enzymes of this kind it is possible to add additional insertion sites per plasmid.
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For our construct we designed a cloning site which when digested with Bsa I will produce Not I like overhang and Spe I like overhang (Fig). Which will ligate to Not I and Spe I but won't be digested after.
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Designing the Bsa I a cloning site that the digestion would result in Not I like overhang and Spe I like overhang flanking the cloning site.(Fig. 3).
<pre>
<pre>
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        Bsa I    Not I'          Spe I'   Bsa I
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Fig. 3
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          Bsa I    Not I'          Spe I'
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5'...GG GGTCTC A^GGCC ….........^CTAG A GAGACC...3'
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          -->
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3'...CC CCAGAG T CCGG^TCCGGCCGCT GATC^T CTCTGG...5'
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5'...GG GGTCTC A^GGCC ….........^CTAG A GAGACC...3'
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3'...CC CCAGAG T CCGG^TCCGGCCGCT GATC^T CTCTGG...5'  
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5'...GG GGTCTC A                CTAG A GAGACC...3'
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5'...GG GGTCTC A                CTAG A GAGACC...3'
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3'...CC CCAGAG T CCGG                T CTCTGG...5'
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3'...CC CCAGAG T CCGG                T CTCTGG...5'
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                                          <--
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                                        Bsa I
</pre>
</pre>
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However there are some limitations Bsa I. Its not an official biobrick restriction enzyme so you have to screen each whole construct for Bsa I recognition sequences. However no worries are needed for inserts. Because only official restriction enzymes treatment is required for them.
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Please be careful Bsa 1 is not official BioBrick enzyme so you must check your plasmid backbone sequence and remove it if there is one . However you don have to worry about insert BioBricks, because they only need to be digested by official BioBrick enzymes.
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Usage standard assembly produces in-frame stop codons in scars. We got around this by using PCR to amplify our inserts. We designed amplification primers to insert mutation and remove both remove change stop codon and Xba I restriction site.  
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For domain fusion, removal of existing stop codons of prefix and/or suffix is essential. This can be easily achieved by designing primers which delete stop codons by adding single point mutation. These primer sets can be used as universal primers which aneal to all BioBricks.
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===Onion===
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==RFC submission==
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We have submitted this method as [[Media:HokkaidoU_BBF_RFC_87.pdf|BBF RFC 87]].
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For more details about RFC submission, please see also [[Team:HokkaidoU_Japan/Project/RFC87|here]].
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Latest revision as of 12:38, 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 Bsa I a cloning site that the digestion would result in Not I like overhang and Spe I like overhang flanking the cloning 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 be careful Bsa 1 is not official BioBrick enzyme so you must check your plasmid backbone sequence and remove it if there is one . However you don have to worry about insert BioBricks, because they only need to be digested by official BioBrick enzymes.

For domain fusion, removal of existing stop codons of prefix and/or suffix is essential. This can be easily achieved by designing primers which delete stop codons by adding single point mutation. These primer sets can be used as universal primers which aneal to all BioBricks.

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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