Team:Northern BC/Results
Results
Build RFC-10 Compatible Yeast Shuttle Vectors
We have chosen to build our opioid sensor in the yeast Saccharomyces cerevisiae in order to take advantage of already existing intracellular machinery that we intend to hijack for amplification of the signal that is generated when an opioid molecule binds our detector protein. In order to successfully express our biosensor components, we required several yeast shuttle vectors that were able to be propagated in E. coli, but our sensor components expressed in S. cerevisiae. These were very generously donated to us by Dr. Stephen Rader (UNBC), along with the yeast strain BY4741.
BY4741 is an S288C-derived laboratory strain of S. cerevisiae in which there is a partial or complete deletion of the open reading frame of various genes required to produce certain amino acids (Table 1). In order for the yeast to survive, they must be supplemented with these amino acids in their growth media. Alternatively, the gene for specific enzymes that are required to produce the amino acid can be incorporated into a yeast vector to act as an auxotrophic marker. Expressing our sensor components from shuttle vectors that also express these auxotrophic markers allows us to track uptake of our genes by yeast cells grown in media that lacks these amino acids. The auxotrophic markers we chose to use were HIS3 and LEU2, expressed from pRS423 and pRS425, respectively. Both of these shuttle vectors contain a gene whose expression confers resistance to Ampicillin to allow for selective propagation of these vectors in E. coli (Table 1).
Site-directed mutagenesis of the yeast shuttle vectors
pRS423 and pRS425 are commonly used in laboratories as yeast shuttle vectors, and therefore, we chose to use these as a starting point to build our opioid biosensor. However, both of these vectors contain restriction enzyme sites in regions outside of the multiple cloning site that are part of the RFC-10 biobrick prefix and suffix (Figure 1). In order to make these vector RFC-10 compatible, we had to first remove these extra restriction enzyme sites, and we chose to do this by sequential site-direct mutagenesis. The first step was to design a forward and a reverse primer that overlapped the region we intended to mutate, with each of these containing a single base change that destroyed the restriction enzyme recognition sequence, but did not alter the amino acid coded for. We successfully removed both a PstI and XbaI site from pRS423, and an EcoRI and XbaI site from pRS425.
To verify our mutations were present and that no other mutations had been unintentionally made during our site-directed mutagenesis, we provided the Northern Analytical Laboratories Services Genetics Facility with our sample DNA and Ms. Caitlin Pitt kindly ran the sequencing experiments for us. Sequencing results confirmed that all mutations were indeed present. Going forward we named our new pRS423 vector ‘pGEM13’ and our new pRS425 vector ‘pGEM18’.
Addition of the RFC-10-compatible biobrick multiple cloning site into the yeast shuttle vector
In order to both insert our biosensor ‘detector’ gene into the pGEM13 backbone, as well as to create a yeast shuttle vector that was biobrick-compatible for parts insertion and characterization, we needed to add the biobrick prefix and suffix to the multiple cloning site of the shuttle vector. To do this, we ordered a fragment of DNA containing the biobrick prefix and suffix and inserted it into the EcoRI-SacI site of pGEM13 while at the same time maintaining as many of the shuttle vector multiple cloning sites as possible (Figure 2). The successful incorporation of the DNA fragment was confirmed by sequencing through the multiple cloning site with the M13 sequencing primers.
Effect of Methanol Addition on BY4741 Cell Growth
Since the opioid and non-opioid drug samples we intend to test in our system are all suspended in methanol by the chemical supplier, we had to make sure methanol was not going to kill our cells. To this end, we tested the effect of increasing amounts of methanol on the growth rate of BY4741 cells (Figure 3). We found that as long as the methanol concentration is kept below 1% we should not see considerable adverse effects when we test our biosensor with these drug samples.
