Week 1 - Plasmids

Question 1

List some cloning vectors

Answer 1

Plasmid, phage and viruses

Question 2

What are cloning hosts

Answer 2

They are organisms that are compatible with vectors, they are often specific to vector and are usually auxotrophic

Question 3

Name some commonly used E. coli strains for cloning

Answer 3

BL21, HB101 and XL1-Blue

Question 4

Compare and contrast Genotype vs Phenotype

Answer 4

The genotype is the gene content (Allele or mutation), while the Phenotype is the observable characteristics of the genotype

Question 5

What are cloning factors derived from?

Answer 5

natural moblie genetic elements such as plasmids, transposons, phages or viruses.

Question 6

key features needed for a cloning vectors

Answer 6

Need to modifiable, selectable, transferable and detectable

Question 7

Explain plasmid in compatibility

Answer 7

Plasmids of the same Ori type cannot coexist in the same cell, one plasmid will be lost during replication, While plasmids of differing Ori replications can coexist

Question 8

list some general requirements needed for plasmid cloning vectors

Answer 8

Easily introduced to the cell, can replicate and maintain cell pop in cells, must be selectable, identifiable, nucleotide sites to insert target DNA

Question 9

List ways of introducing DNA into host cells

Answer 9

Conjugation - some plasmids
Transformation - plasmids
Transduction - Phage’s and viruses
Transfection - eukaryotic cells only

Question 10

What is conjugation and what vectors can do it?

Answer 10

Only some plasmids can perform conjugation, with it being a method of transferring genes to another cell. It can be used to transfer more than just plasmid DNA, some other plasmids in the cell can hitch a ride.

Question 11

What is Transformation and why is competent important?

Answer 11

It is the uptake of free or naked DNA into the cell, bacteria need to competent to take up the DNA. Competence is state which involves specialised mechanisms for DNA uptake

Question 12

E. coli is not naturally competent but it is the main cloning host used why?

Answer 12

We know a lot about it, such as its phenotype characteristics, and how to control DNA inside its cells

E. coli host strains have antibiotic resistances to aid in selection, often taking advantage of operon systems to prevent or activate growth

Question 13

How can E. coli be made competent

Answer 13

2 main methods:
Calcium chloride treatment via heat shock
And Electroporation, the electrical charge disrupts the cell membrane allowing DNA to enter