In a 2015 paper in Nature Chemical Biology about engineering the early steps of

Question

In a 2015 paper in Nature Chemical Biology about engineering the early steps of the morphine pathway into yeast, Dueber and coworkers built a library of 200,000 CYP76AD1 mutants in order to enhance enzyme-catalyzed conversion of tyrosine to L-DOPA (the first step in the morphine pathway) and also limit a second oxidation also catalyzed by the WT enzyme that converts L-DOPA to the L-Dopaquinone (en route to melanin - see figure below). The paper is posted on canvas, but is not required to answer the questions. L-Dopaquinone? Melanin to L-Tyrosine L-DOPA Betaxanthin DODC BIAS (eg, morphine) Dopamine

Explanation / Answer

The error prone PCR are generated with mutated and regulated Taq polymerase which lack the proof reading capacity on the growing chain of DNA. The lack of DNA proof reading by Taq polymerase enzyme the mutation in the DNA stand can be regulated and desired mutant can be inserted at the desired location in the DNA strand and these DNA strand can be inserted in to suitable vector after which this vector can be in inserted in the plasmid and clone to produced the colonies on the Petri plate. We can observe the mutated colonies on petri plate by adding selective medium and antibiotics which allows the growth of selected microorganism and if the mutation is inserted in the desired location no colonies will grow which will confirm the error prone PCR is done or not.      

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