L3 Vectors

Question 1

What are vectors

Answer 1

Plasmids
Bacteriophages
Cosmids
Artificial chromosomes

Act as a molecule carrier for foreign DNA and ensures PROPAGATION and REPLICATION within the host cell

Modules providing essential requirements for cloning

Based on natural occuring DNA

Cloning vector: used to reproduce or amplify a DNA sequence

Question 2

Plasmid vectors

Answer 2

Contain at least 1 replication origin and can be replicated within appropriate host

Code for phenotypic trait that can be used to detect their presence, allows differentiation of untransformed cells

Multicloning site has multiple resitriction enzyme, allowing cloning of different DNA fragments

Question 3

Plasmid classification

Answer 3

Based on:
Copy number (low/high)
Compatability
Host range - limited replication host

Overcoming host range limitation by using SHUTTLE VECTOR e.g E.coli and yeast

INTEGRATIVE VECTOR: cannot replicate in certain hosts. produce single copy of gene.

Question 4

Selection of cells containing plasmids

Answer 4

Insertional inactivation: The cloned insert disrupts a gene in the plasmid, causing inactivation of the gene.

process: vector ligation > insertion into plasmid > inactive gene (recombinant plasmid)

Question 5

Disadvantages cloning with plasmid

Answer 5

Transformation efficiency is very low <1%

Transformation with large plasmids is very inefficient.

Question 6

Bacteriophage vectors

Answer 6

Infection of E.coli
Phage DNA can only be packaged into the virus if viral size is 78-105%

DNA must contain cos sites

Must use precise genome size

There are 2 pathways when using this method.

Lysogenic: Where the lambda DNA replicates
OR
Lytic: where the lambda DNA induces viral protein replication.

Question 7

Propagation of lambda (viral vector)

Answer 7

Phage kept at 4 degrees, allows stability for months

Bacteria susceptible to phage infection in maltose medium.

These 2 solutions are mixed and poured into an agar medium

Question 8

Advantages of using phages

Answer 8

Well understood
Reliable infection of E.coli, must better than plasmid.

The phage gene encoding for Lytic process is removed.

Question 9

Cosmids

Answer 9

A plasmid that carries a copy of the cos sequence required to package DNA into Phage lambda particles.

Cosmids behaves like plasmid hence they have good compatability

Question 10

Artificial chromosomes

Answer 10

Bacterial artificial chromosomes (BAC)

• They are engineered plasmids
• Contain F factor origin of replication
• 200 kb insert size

Eukaryotic Artificial chromosomes
-Must contain elements required for chromosome stability and function (telo/centromeres)

Yeast (YAC)

• 500 kb cloning
• Carries gene for selection in yeast

Human (HAC)
-Exogenus chromosome created de novo from cloned, structurally defined elements. Basically a functioning centromere, potent in gene therapy.