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Q10 - Yeast Secretion Trap Screen for Phytopathogen Secretomes

Theoretical 2 Real exam question - full text reproduced under IBO's CC BY-NC-SA 4.0 license
Recombinant DNA & Biotechnology Techniques

Phytopathogens use their secreted proteins “secretomes” to attack plant hosts. The yeast secretion trap (YST) functional screen is a method used for isolation and identification of these secreted proteins. This method involves generating a vector library that includes cDNAs (containing 5’ non-coding sequences) synthesized using phytopathogen RNAs fused to a mutated form of the Saccharomyces cerevisiae suc2 reporter gene. Suc2 encodes invertase which is the only protein used by yeast for sucrose degradation. Degradation occurs in the extra-cellular medium. The mutated form of the gene suc2-SP encodes an invertase that lacks the signal peptide for secretion at the amino terminal. The fusion library is used to transfect an invertase-deficient yeast strain, and the cells are subsequently plated on a sucrose selection medium. Each rescued cell is expected to contain a recombinant vector whose cDNA that encodes a signal peptide for the chimeric protein. Vectors are isolated and their cDNAs are sequenced to identify the secreted proteins. Figure 1 is a schematic presentation of the vector, and Figure 2 summarizes the protocol described above.

GAATTC: EcoR1 recognition site; GCGGCCGC: Not1 recognition site GAATTC: EcoR1 recognition site; GCGGCCGC: Not1 recognition site

Using the information and data, determine which of the statements are true or which are false.

A. For the purpose of this screen, oligo-dT primers can be used for synthesis of cDNAs.
B. Variable number of Cytidine nucleotide(s) after the Not1 site in the vector ensures achieving a correct reading frame.
C. Presence of signal peptide in the fusion protein will not necessarily ensure the growth of yeast on the selection medium.
D. For introduction of EcoRI and NotI sites to ends of cDNA molecules, linkers (which are short double stranded oligonucleotides; example shown below) are better than adapters (which have single stranded ends; example shown below).
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Question reproduced from IBO 2018, Theoretical Paper 2, licensed under CC BY-NC-SA 4.0 - attributed to the International Biology Olympiad. Open the full exam PDF · Community solutions (unofficial)