Describe in vitro compartmentalisation used in directed evolution experiments. What are the advantages of using this technique over in vivo experiments?

Advanced Biotechnology

Instructions

– Maximum of 500 words for each question

– write a well-structured answer in a series of paragraphs answering points asked in the question. The answer should correctly use the appropriate terminology and referencing.

– When you address the question, you should interpret and discuss the data provided.

– You should also use relevant literature to add a different perspective and/or more detail.

– Include diagrams where appropriate, but these must be referred to in the text.

– Element of criticality and synthesis are a must.

– A well-founded conclusion should also be included

Questions to be answered

Question 1. Describe in detail the pathways for engineering functional CO2 -concentrating mechanisms into plant chloroplasts.

Question 2. This question is in two parts, each carrying equal marks.

(a) The formal definitions for metabolite identifications have been defined by the Metabolite Standards Initiative.  With particular reference to the identification of tryptophan using GC-MS discuss how Level 2 identification is achieved and how this can be improved to Level 1.

(b) Now that you know you can detect tryptophan with GC-MS; how can its level in a yeast culture be quantified?

Question 3. This question involves genetic engineering and is in three parts, each carrying equal

marks.

(a) Describe the principles of the ‘maquette’ approach to de novo protein design.

(b) Describe in vitro compartmentalisation used in directed evolution experiments. What are the advantages of using this technique over in vivo experiments?

(c) What is the difference between non-homologous end-joining and homology-directed repair of a nuclease-induced double-strand break? How can each be used in genome editing to introduce mutations?

Question 5. This question involves genetic modification and is in three parts, each

carrying equal marks.

(a) What are the differences between rational design and directed evolution approaches to protein design?

(b) You want to introduce an indel into a gene encoding a eukaryotic protein of known structure to disrupt the gene’s function. Where in the gene sequence should you target, and why?

(c) How does off-target nuclease activity with CRISPR-Cas9 occur? Why is this a problem? How is this most-commonly minimised?

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