Week 5 Tuesday Flashcards
(25 cards)
What are the steps of the biochemical production process?
Strain generation &screening, Scale-up, Production, downstream processing
What is the importance of the strain generation & screening step?
Screens as many syn bio systems as possible and ensures you get the best one to scale up before you spend oney
How many genes does the plasmid have?
4, crtI. crtYB, crtE. tHMG1
What are the three main ways to optimize our strain? (general list)
Strain robustness, metabolic flux, pathway optimization
What does strain robustness mean?
better growth and more stability (maintaining its pathway)
What does increasing metabolic flux mean?
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.
What does pathway optimization entail?
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.
What are some pros of integrating the beta carotene into the genome instead of the plasmid?
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
What are some cons of integrating the beta carotene into the genome instead of the plasmid?
Much more difficult. You have to be careful to not interrupt the expression of another gene that is important for cell growth
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
True
What are two ways genes are integrated into the genome (just name them)?
HDR and NHEJ
What is HDR?
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
What is NHEJ?
Non-homologous end joining: easily stitches DNA right back together, usually by removing or adding a nucleotide (random).
What are the pros and cons of HDR?
HDR is more accurate but slower
What are the pros and cons of NHEJ?
is faster but less accurate
Do animal cells like HDR or NHEJ?
NHEJ
Do plant cells like HDR or NHEJ?
NHEJ
Do yeast cells like HDR or NHEJ?
both but HDR is less efficient
Do bacteria cells like HDR or NHEJ?
HDR for most bacteria but some are able to use NHEJ
Which repair method is better to harness for genome editing: HDR or NHEJ?
- 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
Why do most cells wat to do NHEJ?
because it is less extensive and requires less energy from the cell, also quicker
How does inactivating a gene with NHEJ work and what are some complications?
- 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.
How does inactivating a gene with HDR work and what are some complications?
- 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.
____ is quicker and more efficient but leads to non-specific edits, while ____ is slower and less efficient but generates specific edits.
NHEJ, HDR
