Overview

As part of the 2017 iGEM competition, we aimed to create a shuttle vector that could be expressed in both E. coli and Pichia pastoris, so that other iGEM teams could design plasmids for use in both of these organisms. We wanted the yeast selection marker (which makes the plasmid a shuttle vector) to be located within the backbone of the plasmid. In other words, so that other teams could easily put genes into the shuttle vector plasmid, we did not want the yeast selection marker to be within the prefix/suffix regions of psB1C3. This led us to design psB1C3mut, which has blunt-end restriction sites for cloning not within the prefix/suffix region of psB1C3.

The iGEM registry uses part BBa_J04450 as a template for PCR to create the psB1C3 backbone, which is sent out to all of the iGEM teams. To make BBa_K2428000, the UChicago team simply used the linearized psB1C3 backbone sent from iGEM headquarters. Our part is a mutagenized form of the backbone psB1C3. The UChicago team cut the linearized psB1C3 sent from iGEM with XbaI and SpeI, then ligated the DNA back together to create a circularized psB1C3 plasmid. QuickChange PCR was performed to create blunt-end restriction sites (such as HpaI) for cloning not within the prefix/suffix region of the original psB1C3 iGEM plasmid. The resulting part we created is BBa_K2428000.

We cut the psB1C3mut we made with HpaI and EcoRI as part of a diagnostic digest. We ran a gel of these diagnostic digests and took an image of the following result: This gel shows that the psB1C3mut was cut at HpaI and EcoRI into a 1870 bp region and a 200 bp region. This indicates we successfully mutagenized psB1C3 into psB1C3mut due to the addition of the HpaI cut site.

We transformed psB1C3mut into E. coli and grew the bacteria in LB + chloramphenicol plates, since psB1C3mut has chloramphenicol resistance. Colonies grew on the plates, demonstrating that the psB1C3mut retained chloramphenicol resistance from psB1C3.

The lab we work in provided us with a plasmid, pOW1, which contains SCARG4 (a gene that produces arginine) and PARS2 (an autonomous replicating element). We put SCARG4 and PARS2 into psB1C3mut to create the plasmid psD000.

This second part (BBa_K2428001) contains one of our other parts (BBa_K2428000, which when circularized is psB1C3mut), SCARG4 (a gene that produces arginine) and PARS2 (an autonomous replicating element). We PCRd the region containing these two genes and performed gel electrophoresis of the DNA, which confirm that we had successfully amplified these genes (2619 bp):

Using Gibson assembly, we added these two genes into psB1C3mut, resulting in psD000 (a plasmid containing the part BBa_K2428001). The presence of SCARG4 and PARS2 in our construct means that psD000 is a shuttle vector (it can be transformed and expressed into both E. coli and yeast, since the DNA has autonomous replicating element and self-producing arginine).

We transformed psD000 into E. coli and grew the bacteria on LB + chloramphenicol plates, confirming psD000 can be expressed in bacteria and has chloramphenicol resistance:

We transformed psD000 into the yeast strain Pichia pastoris and grew the yeast on -Arg dropout plates. This confirmed that psD000 can be expressed in yeast and has the ArgArs gene (which produces arginine, allowing the yeast to survive on plates not containing arginine).