Part:BBa_K782057

SacI/SpeI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors

All ccdb parts have been withdrawn from the Registry. Samples of parts containing the ccdB gene cannot be requested. - iGEM HQ

We have designed a new system for cloning of genes with non-standard restriction sites at 5' and 3' ends into BioBrick vectors. Before our system was introduced, cloning a gene from a non BioBrick vector with a different multi cloning site into a BioBrick vector with the standardized BioBrick cloning site was time consuming and expensive. If we wanted to clone a gene that was in a vector with noncompatible restriction sites into a BioBrick vector we previously had to perform a PCR reaction with which we would add overhangs with appropriate restriction sites. We would then have to digest our PCR product with restriction enzymes and ligate it into a BioBrick vector.

With the system we designed, it is now possible to digest your gene from almost any plasmid with non-standard multi cloning site with several combinations of restriction enzymes and then insert it into a BioBrick vector using just a ligation reaction. We can therefore skip the problematic, time consuming and nonreliable PCR reaction of the gene and thus avoid the possibility of new point mutations in the product.

The strategy is based on the type II restriction enzyme BsaI. Target vectors are composed of standard BioBrick restriction sites and a ccdB expression cassette. In the process of preparing the target vectors, ccdB was cloned into the vector together with a kanamycin resistance marker in order to simplify the cloning, but kanamycin resistance has no significant role in the final constructs. CcdB-KanR expression cassette is flanked with BsaI restriction sites. After BsaI restriction the target vector is digested into backbone with the standard BioBrick restriction sites and any desired 4 nucleotide overhang. That means that any fragment can be cloned in-between the standard BioBrick restriction sites. Both cutting sites of BsaI restriction enzyme are designed in a way that the original restriction sites flanking the insert in the original vector are never reconstituted. So someone can even use a BioBrick restriction enzyme for subcloning of desired genes into the BioBrick standard vector in this way.

Figure 1. Scheme representing the new system for subcloning of non standard genes into a BioBrick vector. Using a series of vectors that we have prepared and deposited to the Registry, someone can subclone desired genes from almost any available vector to the BioBrick standard vector pSB1C3.

SacI overhang can not be produced with BsaI restriction enzyme. This part should be digested with SacI and with BsaI.

We have designed 16 different multicloning sites for »BioBrickization« of genes flanked with combinations of restriction enzyme sites listed below:

EcoRI/BamHI; EcoRI/XbaI; EcoRI/SacI; HindIII/BamHI; HindIII/XbaI; HindIII/XhoI; HindIII/SacI; BamHI/XbaI; BamHI/XhoI; BamHI/SacI; XbaI/BamHI; XbaI/XhoI; XbaI/SacI; SacI/BamHI; SacI/SpeI; SacI/XhoI.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 646
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 552
  Illegal BsaI site found at 1489