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 to screen vast libraries of proteins. It 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.

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. Also 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 was the cheapest.

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.

And because only backbone has to be digested by Bsa I you don't have to worry about inserts having Bsa I sites.

We designed a cloning site which when digested with Bsa I will produce Not I like overhang and Spe I like overhang. 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'

We dealt with TAG stop codon at Xba I site by inserting a mutation and destroying it. Relatively easy step using just primers and PCR.

New BioBrick standard would not by realistic as making several cloning sites with the same restriction site would defeat the purpose. Using Bsa I Cloning Site as a BioBrick will open the door to insert new BioBricks in various places of the construct. Using other Bsa I like restriction sites you can create multiple insertion sites in the construct.

Onion