Team:HokkaidoU Japan/Project/Backbone

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=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 reporter, Bsa I Cloning Site. Desired protein are 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 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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Last year we used T3SS to inject GFP to mammalian cells. This year we wanted to explore T3SS limits. We submitted it to a injectable protein screening. We made a small library of proteins with distinguished structures which were chosen from 2011 Biobrick distribution.
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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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Assembling each protein, injection signal and reporter would have been a laborious task which we didn`t want to endure. So ready-to-inject backbones was proposed(Fig.1). 
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We dsigned Bsa I Cloning Site to facilitate quick assembly of proteins to inject. Thus it`s name ready-to-inject backbone. All inserts must be PCRed with specific primer to remove stop codon. Of course all inserts will be in-frame. See here for details.
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Another problem was how check if protein was injected. To solve it we used a distinct property of Glycogen
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Synthase Kinase 3 β, it is phosphorylated only in eucaryotic cells. This phosphorylation can by detected with phospho-specific antibodies. Thus we now can easily detect if protein was injected in eucaryotic cells. This property is exhibited by first 13aa of this protein<sup>[[#References|[1]]]</sup> which makes it very small reporter. See here for details.
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This design is one of the simpler ones. SlrP is an injection signal, without it the protein cannot be secreted. GSK is a reporter, by detecting phosphorylation of it you can distinguish whether it has been it eukaryotic cell. For us it is an evidence of successful injection. Bsa I Cloning Site a neat cloning site which can be used for inserting various biobrick while retaining the whole constructs BioBrick properties. It is discussed bellow
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=Bsa I Cloning Site=
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[[File:HokkaidoU_Japan_2011_Bsa I_cloning_site_icon.png|left|50px]]
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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 to screen vast libraries of proteins. It designed that inserted biobrick would be fused to preceding signals.
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Bsa I restriction enzyme is in distinguish group of enzyme which cutting site is different from recognition site. Unlike EcoR1 or Pst1, 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).
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<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 Ecor1 digested strand. Also long as NAATTN won`t become GAATTG it wouldn`t not be digested by Ecor1 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 was the cheapest.  
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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).
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However there are some limitations Bsa I is not an official biobrick Restriction enzyme so you have to screen each part for Bsa I recognition sequences. pSB1A3 has one in Ar locus which requires silent mutation or avoiding using it. Thus fur we didn`t encounter other BioBricks containing it.  
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<pre>
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Fig. 3
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          Bsa I   Not I'          Spe I'
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          -->
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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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And because only backbone has to be digested by Bsa I you don`t have to worry about inserts having Bsa I sites.
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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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                                          <--
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                                        Bsa I
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</pre>
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We designed a cloning site which when digested with Bsa I will produce Not1 like overhang  and Spe1 like overhang. We dealt with TAG stop codon at Xba1 site by insetting a mutation and destroying it. You can find how it is done in protocol section.
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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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=Reporter=
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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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[[File:HokkaidoU_Japan_2011_GSK_reporter_icon.png|left|50px]]
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Glycogen Synthase Kinase 3 β is known to be phosphorylated by several enzymes in eukaryotic cell. We used first 13 amino acid sequence to construct a reporter which phosphorylation state could be detected. 9th amino acid which serine is phosphorylated in eukaryotic cell. There are antibodies which bind to only phosphorylated GSK reporter using them it is possible to distinguish whether it has been it eukaryotic cell. So you can see proteins which were injected into cell and which were not. This was a vital ingredient in our experiments.
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==References==
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==RFC submission==
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# Julie Torruellas Garcia, Franco Ferracci, Michael W. Jackson,1 Sabrina S. Joseph, Isabelle Pattis, Lisa R. W. Plano, Wolfgang Fischer, and Gregory V. Plano. 2006. Measurement of Effector Protein Injection by Type III and Type IV Secretion Systems by Using a 13-Residue Phosphorylatable Glycogen Synthase Kinase Tag. Infect Immun.Vol.74:5645-57. [http://www.ncbi.nlm.nih.gov/pubmed/16988240 PubMed]
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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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