Question

CDNA Library Screen Growth at 23 °C on Growth at 32 °C on media lacking uracil media lacking uracil • Prepared a cDNA library from the wild-type URA3+ cells, which is used to transformed into X cells Each spot represents a single clone growing What are the MOLECULAR differences between clones growing on the two plate? How can these results be used to identify the plasmid containing the wild-type URA3+ gene? Based on these results, how can the identity of gene be determined?

Answer 1

1. Molecular differences: The cells or colonies growing on the first plate get efficiently transformed into X cells and the X cells that might have been deficient in the URA3+ gene , can now synthesise the genes with the help of wild type cDNA transformation and can grow on the plate lacking uracil. On the second plate, the number of transformants are very less which shows that the X cells that were transformed with the URA3+ genes could not take up the cDNA and failed to synthesise uracil and hence did not grow on the uracil lacking medium or maybe the competemce factor of the cells in the second plate was not upto the mark that degraded the transformed DNA and inhibited successful transformation.

2. To study the plasmid containing URA3+ gene, plasmid isolation can be performed using one of the colonies that grew on the plates. Firstly, a pure broth culture of the growing transformed colony can be made and then with the broth, a standard protocol of plasmid isolation can be done and then after the isolation of the plasmid, the resultant band can be further adhered to a radioactive marker of URA3+ and checked to see it's binding affinity.

3. As explained in the last point, URA3+ gene marker can be used to target bind the gene and then through sprctroscopic techniques, they can be identified