Jacob works at Genzymatic, which produces industrial enzymes. He has purified a
ID: 61473 • Letter: J
Question
Jacob works at Genzymatic, which produces industrial enzymes. He has purified a large quantity of amylase from strain 92521 of the yeast Saccharomyces riversidis
3a. How might Jacob clone the gene that encodes this protein? Describe all of the steps.
3b. A group working at a competing firm shows that strain 14853 of S. riversidis makes a great. Assuming that you have access to strain 14853, how can Jacob determine if its amylase gene differs from the gene that he has cloned?
3c. You want to test if the amylase is needed for the growth of S. riversididis on rotting corn stalks. Describe how you might use a gene knockout approach to do this, including all of the steps that would be involved.
Explanation / Answer
3a) Jacob purified the amylase protein from S. riversidis 92521. This purified protein can then be sent for protein sequencing by Edman degradation reaction or by Mass spectrometry.
Once the sequence of the protein is known, then from their the sequence of the gene ( the sequence of the polynucleotide can be derived). For example, if at a particular location the amino acid methionine is present, then its corresponding poly nucleotide sequence will be AUG.
Thus in this way the sequence of the entire gene can be found out.
This gene can now be synthesized in a lab and can be ligated into an appropriate vector, for example YAC vector can be used for cloning in yeastm host.
3b) Now in such a scenario, if the strain 14853 is available, he can find out the sequence of the amylase gene present in this strain. This sequence can now be compared to the sequence 92521 using tools like BLAST.
If there is a 100% match then the 2 sequences are identical. If not, then the 2 sequences differ from each other.
3c) A particular strain of S. riversidis will be required as host which is an amylase auxotroph( amylase-). This means that this particular strain will not produce amylase and amylase has to be given externally for growth. This is a gene knockout for amylase.
Now, two sets of experiments are run simultaneously one of which is a control and the other is the experimental setup.
The control contains amylase+ strain that is grown in a medium of rotting corn stalks while the experimental setup contains the amylse- strain growing on a medium of rotting corn stalks. Now if both the setups show growth of the organism, it indicates that the organism can grow in the absence of amylse.
While if the experimental setup shows no growth, it means that amylase is necessary for growth in such a setup.
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