Dry Lab

Figure 1. Basal vectors of the pMNBB P. pastoris expression system. Note that the basal vectors do not contain the oriT sequence that enables conjugation, nor the PARS1 sequence that confers episomal maintenance. Restriction enzymes presented here are entirely unique to ensure compatibility with the BioBrick™ platform.

   Our approach for assembling these backbones was based on PCR amplification and Gibson assembly rather than a restriction enzyme-mediated cloning strategy. The sequences for pMNBB-ICI and pMNBB-CCI were divided into four and five pieces, respectively. The first 20 bp of the 5’ end of each partition contains the final 20 bp of the 3’ end of the preceding piece. For example, the first 20 bp of the fourth piece of pMNBB-ICI are identical to the final 20 bp of the 3’ end of the third piece of pMNBB-ICI. These complementary sequences allow pieces to be joined together via overlap extension PCR. Once joined, the vector backbones can be circularized using Gibson assembly (Gibson et al. 2009). Once the basal vectors are created, elements may be swapped using restriction enzymes. For example, the basal, methanol-inducible vector pMNBB-ICI contains the pAOX1 promoter without an internal OriT element. Swapping pAOX1 for pAOX1-OriT would lead to the generation of pMNBB-ITI, a version of the pMNBB vector, which contains a methanol-inducible promoter (pAOX) and can be transferred to P. pastoris using TKC (Figure 2).


Figure 2. Creating the conjugal variant of pMNBB-IXX. (A.) As seen in pMNBB-ICI, the pAOX1 promoter does not contain the OriT sequence. (B.) Addition of the OriT sequence within the AOX1 promoter enables conjugal transfer.

   The project was designed to yield two basal P. pastoris expression vectors, as well as two variations upon the basal vectors, as listed in Table 1.

• Vector Assembly
   We began assembling the vectors by amplifying the individual fragments by PCR using Phusion HF polymerase (New England Biolabs). Next, we attempted several transformations of E. coli C2566 with Gibson assemblies of the vectors. Unfortunately the transformations resulted in either no growth, or growth of unidentifiable colonies, as determined by Go-Green Taq (Promega) colony screens. We believe that the lack of growth may be due to vector not circularizing, or in the case of the unidentifiable colonies that appeared on the pMNBB-CCI plates, limited selectivity imparted by our Zeocin media (25ug/mL).

• Results
   First we investigated the possibility that the vectors were not circularizing. In order to simplify the Gibson assembly we began overlapping fragments, reducing the pieces that would need to join in a successful Gibson assembly. The initial two piece overlaps were successful, however overlaps beyond two pieces were problematic. Gibson assemblies in which the fragment number had been reduced by using two piece overlaps produced the same results as the single piece Gibson assemblies, either no growth, or growth of unidentified colonies. We decided to try an alternate PCR protocol (Shevchuk et al. 2004). Finally we were able to string together the five fragments of pMNBB-ICI.

• Conclusions
   Unfortunately we have not yet been able to assemble either vector in its entirety. The basal inducible vector, pMNBB-ICI, has been visualized in its linear state on a gel. We will continue to work on assembling both vectors so that they may be available for future teams to improve upon and characterize. If the pMNBB system functions as expected, it will certainly be a very useful tool not only for iGEM teams, but for academic research, and industrial processes.

Wet Lab