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 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.

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.

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

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.

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).

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.