Quiz review

Question 1

What are the three reasons Heterologous enzymes often have poor performance?

Answer 1

i.) little or no protein expression (confirmed via protein purification)
ii.) poor substrate affinity (confirmed via LCMS)
iii.)promiscuous substrate activity (confirmed via LCMS)

Question 2

Explain how little or no protein expression can occur and how to fix it.

Answer 2

• Toxic to the cell – change organism
• Bad DNA parts – change promoter, terminator
• Suboptimal codon – codon optimization (certain organisms may prefer certain 3-nt codons)

Question 3

Explain how poor substrate affinity can occur and how to fix it.

Answer 3

• Need more protein – add a second copy of the gene (Adding 2nd copies of pathway genes should help rate-limiting step)
• Need a different protein – metagenomic sourcing (Sourcing enzymes from a multitude of nature’s resources)
• Need a modfied protein– protein engineering (Amino acid sequences can be changed to alter protein function)

Question 4

Explain how promiscuous substrate activity can occur and how to fix it.

Answer 4

• Remove alternate substrate – gene knock-out
• Different protein – metagenomic sourcing
• Modified protein – protein engineering

Question 5

Explain how CRISPR works.`

Answer 5

• CRISPR RNA (crRNA) matches DNA protospacer (or “spacer”) and binds DNA via base-pairing
• Cas9 binds DNA at protospacer adjacent motif (PAM, NGG), unzips DNA, and checks for protospacer match
• Upon match, RuvC and HNH catalytic domains introduce double-stranded break

Question 6

How is CRISPR different from previous methods.

Answer 6

Before you have protein-DNA interactions for CRISPR, RNA and DNA interact. Aka CRISPR RNA.

Question 7

How does Cas9 work?

Answer 7

The CAS9 protein will recognize a really short sequence, aka the PAM, and bind to everyone. It then unzips the sequence, and it will take the CRISPR RNA and see if it base pairs with whatever sequence is next to the pam. If not, it will rezip and move on to the next PAM.