5. Finding Enzyme Genes in a Microbial Consortium (20 points) The rumen of large

Question

5. Finding Enzyme Genes in a Microbial Consortium (20 points) The rumen of large herbivores can be looked at as a continuous flow bioreactor, whereby plant biomass substrate (hay) is eaten by the animal and processed into sugars. A mixture of anaerobic bacteria & anaerobic fungi are responsible for producing enzymes that release these sugars, which can be harnessed for their biotech potential. However, the bacteria & fungi that produce these enzymes are not culturable, and the gene sequences that encode the enzvmes remain unknown 50 m Suppose that you set out to "discover" the enzymes made by the bacteria & fungi found in the cow rumen. To do this, you take a sample of liquid from the cow rumen, and extract DNA & RNA from the microbes found there. You also know that the enzymes increase in population when substrate is abundant. (a.) Can you identify novel enzymes from the DNA sequences alone? Why or why not? (b.) Suppose you are only interested in identifying and studying the enzymes from eukaryotic population of rumen microbes - what features of eukaryotic transcripts could you exploit to separate them from prokaryotic transcripts? How could you do this in practice? (c.) Briefly describe how you could test whether any gene sequences you identify are actually enzymes that break down plant biomas:s

Explanation / Answer

a. No.

We can not simply decipher whether a given DNA sequence can code for a novel enzyme. This is because of multiple steps involved in gene expression. A gene is first transcribed into mRNA. The entire portion does not get transcribed. Again, primary mRNA is processed into mature mRNA by removing introns. The mature mRNA is translated into a protein. Posttranslational modifications may add or remove specific moieties. All these factors affect the activity of the final product.

b. Eukaryotic transcripts contain a 5'-G cap and a 3'-poly A tail. Poly A tail is generally absent in prokaryotic mRNAs (A few prokaryotic mRNAs do not contain a short poly A tail at the 3'-end). So, we can use a poly-T primer (Oligo dT) to specifically amplify eukaryotic genes during reverse transcription step to synthesize cDNA.

c. We can isolate the protein product and perform specific biochemical tests to check whether they can act upon on plant biomass.

For example, we can incubate the protein with cellulose and perform specific tests to determine whether cellulose is converted to glucose.

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