Part:BBa_K1057012

pBAD/araC-BsaI-sfGFP-BsaI

    Gentic switch such as pBAD/araC system is very useful for overexpression of given genes. In order to place the various open reading frames with its RBS under the pBAD/araC system, we improved BBa_I746908 to insert BsaI site in both sides of sfgfp gene. This improvement enables us to use ‘Golden Gate’ cloning Method as described below (Fig. 1):
1) Preparation of insert fragment : Given gene(s) are PCR amplified with the additional sequence coding for ribosome-binding sites and BsaI site.
2) BBa_I746908 and PCR amplified insert fragment is BsaI digested and ligated in a single-pot reaction.
     This method is designed BsaI site doesn't remain on the vector after digesting BsaI. So, you can perform digestion and ligation at the same time. You can obtain desired plasmids in a short time.

Fig. 1 Golden Gate cloning strategy

(A)

(B)
Fig. 2 Efficiency of cloning for gene swapping(sfgfpto mrfp), (A)percentage of recombinant and non-reconbinant clones(N.D. Not Detected)(B)colony fluorescence

1) The highest cloning efficiency (68.9%) was obtained when the insert/vector molar ratio was 1:1. We suppose that the digestion (BsaI) efficiency could decrease with excess amount of insert (resulting in more "non-digested" green fluorescent colonies).
2) We found no non-fluorescent colonies in all tested conditions.
3) When we want to swap sfgfp to another gene with no phenotypic change, we could screen the right clone as non-fluorescent clones.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1206
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1145
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 980
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 962
  Illegal SapI.rc site found at 1250