Part:BBa_K547000

ready-to-inject backbone for T3SS, SlrP taged, with Bsa I cloning site

Fig. 1 Ready-to-inject backbone. SlrP is an T3SS injection signal. Bsa I Cloning Site is used for inserting various BioBrick directly. An fused protein to T3S signal can be expressed under control of constitutive promoter(pTetR).

Last year we made injectable GFP as a reporter of injetion assay. This year, our project started with an aim to conduct direct reprogramming as an application of T3SS.

Since, fusion of signal peptide required for secretion (T3S signal) to each protein would have been a laborious task, development of [http://2011.igem.org/Team:HokkaidoU_Japan/Project ready-to-inject backbone] has been anticipated. To accomplish this, we designed Bsa I Cloning Site and developed plasmid backbone which can facilitate quick assembly of proteins to be injected (Fig. 1).

Bsa I 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.

Verifying data

Fig.4　Control (left) and GFP (right) under blue light excitation

GFP was added into cloning site. It is preceeded by SlrP secretion signal. This results in GFP expression(Fig.4). Which means that proceeding SlrP secretion tag and GFP are fused in frame. This data shows that Bsa I cloning site realy works in practice.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found at 2319
  Illegal suffix found at 137
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2319
  Illegal SpeI site found at 138
  Illegal PstI site found at 152
  Illegal NotI site found at 145
  Illegal NotI site found at 2325
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2319
  Illegal XhoI site found at 1169
  Illegal XhoI site found at 2195
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 2319
  Illegal suffix found at 138
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 2319
  Plasmid lacks a suffix.
  Illegal XbaI site found at 2334
  Illegal SpeI site found at 138
  Illegal PstI site found at 152
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 2991
  Illegal BsaI.rc site found at 2
  Illegal BsaI.rc site found at 3653