HokkaidoU Japan

iGEM 2011 Team of Hokkaido University

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• 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!

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 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.

5'...GGTCTCN^.......3'
3'...CCAGAGNNNNN^...5'

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.

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.

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.

         Bsa I    Not I'           Spe I'   Bsa 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'

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.

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.

Onion