Lecture 24

Question 1

Which vector is based on the F plasmid for large bits of DNA?

Answer 1

BAC Vector

Question 2

What is a replicon?

Answer 2

A nucleic acid molecule of autonomous replication. It needs an origin of replication.

Question 3

What is essential for a replicon?

Answer 3

Chromosomes (needed for growth of cell under normal conditions).

Question 4

What 2 things are not essential for replicons?

Answer 4

Plasmid (they autonomously replicate)

Virus (don’t need an infection to reproduce)

Question 5

What do most plasmid vectors have?

Answer 5

lac-alpha region (with MCS - insertional inactivation)
SP6/T3/T7 bacteriophage promoters by the MCS for RNA transcripts of inserts
Sequencing primers that anneal next to MCS

Question 6

What is an expression vector for?

Answer 6

To make a lot of protein. (E.coli)

Question 7

What does an expression vector require to work?

Answer 7

Plasmid, selectable marker, replication of origin, T7 polymerase gene

Question 8

What is unique about T7 Polymerase?

Answer 8

It’s optimal temp is high so inducible by temperature shift (control). It is also upstream of 6 histidines (easy purification).

Question 9

What vectors are used when cloning larger DNA fragments?

Answer 9

BAC vectors. They can clone 100-150kb fragments. They also have high stability.

Question 10

What does a BAC vector typically need?

Answer 10

F plasmid as it has a low copy number (1-4 per cell).
Replication enzyme, ParA and ParB (partition genes)
Origin of replication

Question 11

How do you make a BAC library?

Answer 11

1. Open BAC vector to make linear
2. Ligate insert with vector plasmid.
3. Transform to E.coli by electroporation
4. Cloning occurs and library forms.