Week 5 Tuesday

Question 1

What are the steps of the biochemical production process?

Answer 1

Strain generation &screening, Scale-up, Production, downstream processing

Question 2

What is the importance of the strain generation & screening step?

Answer 2

Screens as many syn bio systems as possible and ensures you get the best one to scale up before you spend oney

Question 3

How many genes does the plasmid have?

Answer 3

4, crtI. crtYB, crtE. tHMG1

Question 4

What are the three main ways to optimize our strain? (general list)

Answer 4

Strain robustness, metabolic flux, pathway optimization

Question 5

What does strain robustness mean?

Answer 5

better growth and more stability (maintaining its pathway)

Question 6

What does increasing metabolic flux mean?

Answer 6

The cell is consuming its sugar (yeast has a very extensive metabolism), so it takes that sugar which is a carbon source, and it diverts it into all different pathways (some that make amino acids, fats, lipids, etc). We can divert as much of that carbon into our pathway as possible.

Question 7

What does pathway optimization entail?

Answer 7

Different promoter/Terminators. Once its in our pathway, we want to make sure it is working well. Want to make sure we have the proper amount of enzyme for each of those chemical reactions.

Question 8

What are some pros of integrating the beta carotene into the genome instead of the plasmid?

Answer 8

You don’t have to worry about maintaining them or selecting for them. (anything that is on a plasmid you need to select for, i.e., adding an antibiotic or making a different media; both are more expensive). Increases strain robustness

Question 9

What are some cons of integrating the beta carotene into the genome instead of the plasmid?

Answer 9

Much more difficult. You have to be careful to not interrupt the expression of another gene that is important for cell growth

Question 10

True/False: If you decide to take the genes out, it is a lot easier to take it out of a plasmid and not the genome

Answer 10

True

Question 11

What are two ways genes are integrated into the genome (just name them)?

Answer 11

HDR and NHEJ

Question 12

What is HDR?

Answer 12

Homology-directed repair: double-stranded break and the cells will look to see if there is a template that looks just like the template that was broken. If they find homology arms, then they can use that and do recombination to fix the DNA back together

Question 13

What is NHEJ?

Answer 13

Non-homologous end joining: easily stitches DNA right back together, usually by removing or adding a nucleotide (random).

Question 14

What are the pros and cons of HDR?

Answer 14

HDR is more accurate but slower

Question 15

What are the pros and cons of NHEJ?

Answer 15

is faster but less accurate

Question 16

Do animal cells like HDR or NHEJ?

Answer 16

NHEJ

Question 17

Do plant cells like HDR or NHEJ?

Answer 17

NHEJ

Question 18

Do yeast cells like HDR or NHEJ?

Answer 18

both but HDR is less efficient

Question 19

Do bacteria cells like HDR or NHEJ?

Answer 19

HDR for most bacteria but some are able to use NHEJ

Question 20

Which repair method is better to harness for genome editing: HDR or NHEJ?

Answer 20

• HDR is favored because it is more accurate
• NHEJ is faster so if it needs to be done quicker, it will do that one
• If you want to fix a specific mutation, you would want to use HDR because it is more accurate. NHEJ is too random for that scenario

Question 21

Why do most cells wat to do NHEJ?

Answer 21

because it is less extensive and requires less energy from the cell, also quicker

Question 22

How does inactivating a gene with NHEJ work and what are some complications?

Answer 22

• Go in and double-strand break, which allows NHEJ to take over, which would give you a population of different consequences. One cell might randomly add TC and change the amino acids. This wouldn’t necessarily inactivate it, but it would change the gene and translation of the protein.
• You might get some cells that add a T; now the second codon is TAG, which is a stop codon and would inactivate the gene and stop translation. If you use NHEJ you have to select for which cell has the edit you want.

Question 23

How does inactivating a gene with HDR work and what are some complications?

Answer 23

• Go in with a repair template that has homology upstream and homology downstream. You would count on the cell using the template to fix the break. When it does that, it removes the whole gene and does homologous recombination upstream and homologous recombination downstream. It involves making sure you deliver the proper repair template.

Question 24

____ is quicker and more efficient but leads to non-specific edits, while ____ is slower and less efficient but generates specific edits.

Answer 24

NHEJ, HDR

Question 25

How is the double-stranded DNA break created?

Answer 25

CRISPR