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

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.

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.

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