Methods

   Insulin and PCSK1 Open Reading Frame Design Sequences for insulin and its chaperone PCSK1 were obtained through NCBI. Both insulin and PCSK1 were codon optimized for expression in P. pastoris using the online Codon Optimization tool from IDT. The BioBrick consensus sequence was added to both insulin and PCSK1 in preparation for cloning into the shipping vector. Restriction sites that were incompatible with the BioBrick standard, as well as our Pichia pastoris expression system pMNBB were eliminated by altering single nucleotides while maintaining the proper amino acid sequence. Both genes were synthesized by IDT. Insulin was obtained as one 333 bp fragment, while the longer PCSK1 was split into three, roughly 750 bp fragments.

   Fragments for insulin and PCSK1 were amplified by polymerase chain reaction (PCR). Insulin was cloned into the shipping vector. The three PCSK1 fragments were joined into the the full 2300 bp product using overlap extension PCR. The full PCSK1 product was cloned into the shipping vector. Cloning primers were not designed for PCSK1 with flanking BioBrick consensus sequences. PCSK1 was cloned into the pCR®Blunt II-TOPO® vector, provided by Invitrogen.

E. coli C2566 cultures were transformed with PCSK1- pCR®Blunt II-TOPO®. PCSK1- pCR®Blunt II-TOPO® was isolated from transformation cultures and used as a template for PCR amplification of PCSK1 with our insulin primers that contained the BioBrick consensus sequence.

Two transformations were performed using Escherichia coli C2566, one with pSB1C3-Insulin and another with pSB1C3-PCSK1. Both constructs were submitted for sequencing.

Results

The products that were cloned into the shipping vector were verified by gel electrophoresis (Figure 1).

Several colonies were identified in colony screens for PCSK1 and insulin in transformants containing the shipping vector. Seen below are verification gels for PCSK1 and insulin.


Figure 1. PCR colony screen of PCSK1-pSB1C3 transformants. Note the banding in each lane at roughly 750 bp, consistent with the second fragment of PCSK1.


Figure 2. PCR colony screen of Ins-pSB1C3 transformants. Banding can be seen at roughly 333 bp, consistent with the full insulin ORF. Sequencing data received for both PCSK1 and insulin was inconclusive.

Parts List
BBa_K1187001   Human insulin, codon optimized for expression in P. pastoris