HokkaidoU Japan

iGEM 2011 Team of Hokkaido University

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.