The proinsulin gene was positively detected in our transformed algae using genotyping PCR
We were finally able to detect the algae proinsulin gene. We had re-cultured the emergent transformed microalgal colonies on minimal media to ensure that the colonies contained our pASapI-proinsulin DNA vector. Only those microalgae which contain our DNA construct (which also contained a restored photosytem gene) could fix carbon dioxide and survive. Surely enough, the colony survived the re-platings and continued to grow well.
Several months later, we designed DNA primers that would amplify the insulin gene if it was successfully integrated into the microalgal chloroplast.
We designed two primers that amplified out a PCR product with an expected size of 486 basepairs.
The following genotyping primers were synthesized and used in a 50uL PCR reaction.
First we needed to extract the genomic DNA. We used ethanol, boiling water, and Chelex 100 to lyse the cells and purify the DNA. Adding Chelex helps to prevent the genomic DNA samples from being degraded by removing metal ions which may interfere with the PCR annealing process or might catalyze enzymes that facilitate the breakdown of genomic and chloroplast DNA.
We then ran the polymerase chain reaction and loaded the product onto an agarose gel containing a fluorescent DNA stain. A positive PCR band for the proinsulin gene was observed, which matched the expected size of 486 nucleotide basepairs.
Next, we are planning to conduct an immunoblot to try and quantify the amount of insulin that the gene is producing. We are hoping to quantify a detectable level of expression, which will be our next challenge.
Thank you to all the backers for being patient while we continued to work on the technical deliverables. Science is 90% troubleshooting and 10% success.