Week 4 Tuesday Flashcards
(22 cards)
What are the steps of plasmid production and delivery?
DNA assembly aka cloning, propagation in E. coli, Plasmid extraction, Delivery to host (S. cerevisiae)
How are novel DNA constructs assembled?
Through nature or manufactured synthetically
What is a con of Manufacturing synthetically?
You can usually capped around 200 bases
What is an oligo?
A stretch of nucleotides
What 4 things can you do with a DNA template?
Cut, Amplify, Stitch, and Replicate
How does cutting a DNA template work?
recognizes the short DNA sequence (top and bottom), binds to and cuts both strands of dsDNA leaving a staggered top or blunt bottom ends
What are the origins of Endonuclease?
bacteria immune systems
What is a motif?
A short DNA sequence withing a longer piece of DNA
What is the enzyme for amplifying?
DNA polymerase
What are the steps of PCR?
- dsDNA is dentaured using heat to produce ssDNA
- Primers anneal to the ssDNA
- A heat-resistant polymerase extends the ds DNA
What is the purpose of PCR?
to amplify
What is the enzyme for stitching DNA?
DNA ligase, recombinase
Explain what happens during stitching DNA?
-Catalyzes phosphodiester binds between free phosphate ( 5’) and free hydroxyl (3’) ends of DNA
- Ligates “blunt ends” or complementary “sticky ends”
What are ligase origins?
DNA replication and repair
Why don’t we use linear DNA for our final constructs?
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.
How do we ensure the cell will replicate our plasmid?
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
What are the advantages and disadvantages of using E. coli as a host?
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.
What is the enzyme for cutting DNA?
Restriction endonuclease
What is the enzyme for replicating DNA?
DNA polymerase
Describe Gibson Assembly.
-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.
Explain Golden Gate assembly.
- 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.
