Week 4 Tuesday

Question 1

What are the steps of plasmid production and delivery?

Answer 1

DNA assembly aka cloning, propagation in E. coli, Plasmid extraction, Delivery to host (S. cerevisiae)

Question 2

How are novel DNA constructs assembled?

Answer 2

Through nature or manufactured synthetically

Question 3

What is a con of Manufacturing synthetically?

Answer 3

You can usually capped around 200 bases

Question 4

What is an oligo?

Answer 4

A stretch of nucleotides

Question 5

What 4 things can you do with a DNA template?

Answer 5

Cut, Amplify, Stitch, and Replicate

Question 6

How does cutting a DNA template work?

Answer 6

recognizes the short DNA sequence (top and bottom), binds to and cuts both strands of dsDNA leaving a staggered top or blunt bottom ends

Question 7

What are the origins of Endonuclease?

Answer 7

bacteria immune systems

Question 8

What is a motif?

Answer 8

A short DNA sequence withing a longer piece of DNA

Question 9

What is the enzyme for amplifying?

Answer 9

DNA polymerase

Question 10

What are the steps of PCR?

Answer 10

1. dsDNA is dentaured using heat to produce ssDNA
2. Primers anneal to the ssDNA
3. A heat-resistant polymerase extends the ds DNA

Question 11

What is the purpose of PCR?

Answer 11

to amplify

Question 12

What is the enzyme for stitching DNA?

Answer 12

DNA ligase, recombinase

Question 13

Explain what happens during stitching DNA?

Answer 13

-Catalyzes phosphodiester binds between free phosphate ( 5’) and free hydroxyl (3’) ends of DNA
- Ligates “blunt ends” or complementary “sticky ends”

Question 14

What are ligase origins?

Answer 14

DNA replication and repair

Question 15

Why don’t we use linear DNA for our final constructs?

Answer 15

Circular DNA is more stable than linear DNA because it doesn’t have loose ends that are prone to degrading by nucleases. Circular DNA is typically more efficiently taken up by cells during transformation processes.

Question 16

How do we ensure the cell will replicate our plasmid?

Answer 16

Incorporate a selection marker so that you can make sure you are only growing cells with the plasmid. Ensure a suitable origin of replication. Correct phase of growth, add a reagent to throw off host defense system, add chemicals to neutralize

Question 17

What are the advantages and disadvantages of using E. coli as a host?

Answer 17

Now you need to be able to replicate in 2 cell types. It makes your plasmids bigger. If you are not careful, you might make something toxic to E. coli. The advantage is that it grows quickly, easily grows, and has well characterized parts.

Question 18

What is the enzyme for cutting DNA?

Answer 18

Restriction endonuclease

Question 19

What is the enzyme for replicating DNA?

Answer 19

DNA polymerase

Question 20

Describe Gibson Assembly.

Answer 20

-Involves the use of overlapping sequences at the ends of the DNA fragments. A single reaction combines a 5’ exonuclease, a DNA polymerase, and a DNA ligase, allowing the overlapping ends to anneal and be ligated.
-Can join multiple fragments simultaneously in a single step and is less dependent on restriction sites, making it more flexible for assembling complex constructs.

Question 21

Explain Golden Gate assembly.

Answer 21

• Utilizes Type IIs restriction enzymes that cut outside their recognition sites, creating sticky ends. Fragments are assembled by mixing together with these enzymes, allowing for the directional insertion of DNA pieces.
• Facilitates the precise and ordered assembly of multiple fragments in a one-pot reaction, and the use of unique restriction sites allows for easy and efficient cloning.