Please Explain the Essential Components of a Plasmid? and explain the following
ID: 260464 • Letter: P
Question
Please Explain the Essential Components of a Plasmid? and explain the following problem:
Alex wants to reintroduce the ability to synthesize lysine into a bacteria population of that cannot synthesize lysine using a plasmid carrying LYS gene.
To select against the bacteria that did not take in the plasmid, what kind of selective media does Alex need to use?
One day after transfection, no visible colony grows. Aaron suggests that Alex might have accidentally added Neomycin which kills the bacteria. Alex insists that he would never make such an absurd mistake. How can Alex show that he used the correct selective media (no neomycin added)?
What might have gone wrong in Alex’s experiment?
Please try to explain it thoroughly so I can understand the concept! Thank you so much in advance!
Explanation / Answer
A plasmid is closed circular DNA molecule that can propagated in a suitable host cell independent of its genomic DNA.
Common features of a plasmid:
i. Ori: Replication and maintainance
ii. rop: Genes involved in replication
iii. Selectable marker: Used to screen for positive clones
iv. Multiple cloning site: Contains restriction sites for the insertion of the desired DNA fragment.
In the given case, the LYS biosynthetic gene must be cloned into a suitable plasmid using restriction enzymes and ligase. The ligation mixture should be transformed into the host cell. The transformation mixture must be plated on a suitable selection media.
Here, the parental population is LYS-. So, they can not grow in the absence of LYS.
The plasmid contains LYS biosynthetic gene. So, a true trasformant can synthesize LYS on its own and does not depend upon LYS in the media. hence they can grown in LYS- media.
So, the selection plate must be LYS-.
The plasmid also contains a antibiotic resistance marker such as ampcillin or kanamycin. So, we can also add this antibiotic in the media.
To rule out that a wrong antibiotic is added in the media (for lack growth), plate the cells in different antibiotic containing media.