Part:BBa_K3041014

ipET28

The pET28 plasmid adjusted to the RFC10 biobrick system of iGEM, thereby, allowing for overproduction of proteins. The plasmid contains a thrombin site and a His-tag allowing for His-tag purification of the produced protein

Validation

Not only the type of medium, production strain and other growth conditions influence the protein yield, but can also be influenced by the plasmid used. To create a plasmid optimized for protein production that complies with the biobrick system, iGEM Leiden designed the iGEMized "ipET" plasmid, derived from pET28a(+) (Fig. 1). This plasmid enables optimal recombinant protein production of RFC10 biobricks and His-link purification in E. coli strains. ipET was obtained by adjusting the multiple cloning site (MCS) of pET28a(+) into the prefix and suffix, allowing for direct cloning of your RFC10 biobricks into this high production plasmid. The gene will automatically be placed under control of the T7/lac promoter allowing for induced production after addition of IPTG [0.5 mM - 1 mM]. In order to create the multiple cloning site, two different oligo pairs were designed, which were subsequently annealed and placed into the pET28. Due to the chosen restriction of the pET28 some original features of pET28 were lost, including the ribosome binding site, his-tag and thrombin site. To overcome these problems, the features were added again to the new MCS, resulting in the MSC followed by a His₆-tag, TEV protease cleavage site and the original ribosome binding site. To obtain the new “igemized MCS”, two separate parts were designed, separated by a NcoI site, by the annealing of four short synthesized oligos (Fig. 2). At last, the complete multiple cloning site was placed in the ipET plasmid.

Figure 1. The plasmid map of the adjusted ipET plasmid The map displays all restriction sites of the multiple cloning site, the kanamycin resistance marker and the T7 promoter linked to the Lac operator.

Figure 2. Annealing of multiple cloning sites parts 1 and 2. Lanes 1-4 contain the un-annealed oligos, whereas lanes 6-7 contain the annealed oligos. All samples were loaded onto a 1% agarose gel and ran for 40 min at 100V.

In order to validate the newly obtained plasmid, test digestions were conducted. If linearization of the plasmid could be observed it was concluded the restriction sites were intact. Digestions were performed with EcoRI, XbaI and PstI and they all resulted in linearization of the plasmid (Fig. 3).

Figure 3. Validation of ipET restriction sites Lane 1: undigested plasmid. Lane 2: plasmid linearized by XbaI digestion. Lane 3: ipET linearized by EcoRI digestion. Lane 4: the plasmid linearized by PstI digestion. Lane 5: Ladder.

Furthermore, the plasmid was sent to BaseClear for sequencing using two primers, annealing up- and downstream of the new multiple cloning site. The sequences showed a strong alignment with the theoretical sequence suggesting that the new multiple cloning site was correct (Fig. 4).

Figure 4: Sequence alignment of RFC10 multiple cloning site. Two samples using a forward and reverse primer were sent for sequencing. Both results show a strong alignment with the multiple cloning site.

By creating this plasmid we allow future iGEM teams to achieve upregulation of their protein of interest while following the biobrick system. The plasmid contains several features allowing for successful protein production in E. coli. These include a strong T7-lac promoter enabling strong and inducible transcription of the protein using IPTG as an inducer. The plasmid also contains a His₆-tag, allowing for improved purification using either a copper or nickel-column combined with imidazole wash. In this way, protein production and purification will be fast and accurate. The thrombin-site can be used to cleave the His₆-tag after purification. Of note, this plasmid will only produce in specified E. coli strains, such as BL21 (DE3) or Rosetta, as these strains contain a T7 polymerase required for binding to the promoter.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 168
  Illegal BamHI site found at 58
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 200
  Illegal NgoMIV site found at 1788
  Illegal NgoMIV site found at 1948
  Illegal NgoMIV site found at 4995
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 2868