13. Synthetic Biology 2 Flashcards
What is plasmid ligation?
an enzyme-catalysed reaction which forms permanent covalent bonds between two DNA fragments.
What are ‘sticky ends’?
the two ends of the plasmid formed when both DNA fragments have been digested by the restriction enzyme.
What temperature does ligation happen at and what does this allow?
16°C - low temperatures allow hydrogen bonds to form, which hold the DNA fragment together temporarily.
What is the enzyme used in ligation?
DNA ligase
What does DNA ligase do?
catalyse the formation of new covalent bonds between the 3’ OH groups and the 5’ Phosphate groups on the DNA backbone. These are known as phosphodiester bonds.
What is the cofactor used in ligation?
ATP
What is Religation?
if the same restriction enzyme is used for the initial digest, both ends of the plasmid and insert will have complementary ‘sticky ends’
- this means that the plasmic can stick together without the insert = relegation
Why would we want to avoid using Religation?
it reduces the proportion of recombinant plasmids, thereby also reducing cloning efficiency.
What is the reagent needed to remove the 5’ phosphate groups from the plasmid?
Phosphatase
Why is the insert not treated with phosphatase?
if both the plasmid and the insert were treated, no phosphodiester could form so ligation cannot occur and cloning would fail.
- hydrogen bonds form on the sticky ends
What is other way to prevent religation?
Double digestion
What is Double digestion?
use two different enzymes for the initial digest, so that the two ‘sticky ends’ on the plasmid will be different and religation cannot occur
What does religation mean in regards to the sticky ends?
they will be the same - will have used the same enzyme.
Removal of plasmid
the small chunk of plasmid in between the cut sites will be dissociated as it is no longer attatched. a high concentration of insert increases the chances of it binding to the plasmids ‘sticky ends’.
What is the last step of plasmid ligation?
the insert will be ligated into the plasmid in the correct orientation for the promoter to drive transcription in the correct direction.
