Part:BBa_K1830000

pET15BB

IPTG inducible E. Coli Expression vector with RCF25 compatible MCS

Usage and Biology

pET15BB is a vector designed for expression and purification of proteins of interest from E. Coli. It is derived from the iGEM standard pSB1C3 backbone and so includes a Chloramphenicol resistance selection marker. The LacI operon was added to the backbone for inducible expression as well as a multiple cloning site designed to welcome any RFC25 compatible iGEM part. It include a 5' NgoMIV and a 3' SpeI cut sites for insertion of an RFC25 compatible part. The NdeI and BamHI cut sites of orginal pET vectors have also been conserved for cloning using these more classical restriction enzymes.

Instructions for use

To use this expression vector, inserts must have a 5' NgoMIV cut site and a 3' SpeI or PstI cut site.

Digest the vector and the part with two of these enzymes and ligate the insert into the pET15BB backbone.

Select colonies on 50ng/mL Chloramphenicol media

Induce expression with 1mM IPTG

Purify proteins by nickel gravity column

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3530
  Illegal XbaI site found at 47
  Illegal SpeI site found at 213
  Illegal PstI site found at 288
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3530
  Illegal SpeI site found at 213
  Illegal PstI site found at 288
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3530
  Illegal BglII site found at 3818
  Illegal BamHI site found at 162
  Illegal XhoI site found at 157
  Illegal XhoI site found at 1305
  Illegal XhoI site found at 2197
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3530
  Illegal XbaI site found at 47
  Illegal SpeI site found at 213
  Illegal PstI site found at 288
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3530
  Illegal XbaI site found at 47
  Illegal SpeI site found at 213
  Illegal PstI site found at 288
  Illegal NgoMIV site found at 151
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  

Characterization

The gene coding for E. Coli b-galactosidase was cloned into the expression vector to test its functionality. Expression was induced as described earlier and purification was performed using a gravity nickel column. Two successive elution buffers were used containing repestively 20 and 250mM imidazole to elute proteins bound to the nickel.

Strong b-gal activity was observed in the first fractions eluted with 20mM imidazole buffer, and decreased over the fractions. A significant increase in b-gal activity was also observed in fractions eluted with 250mM imidazole. This increase corresponds to the His-tagged proteins eluted by high concentration of imidazole.

By purifying functional His-tagged b-galactosidase, we were able to prove the functionality of our expression vector for overexpression, His tag fusion and frame conservation in cloning.