Lecture 4 Recombinant DNA Techniques

Question 1

What are plasmids?

Answer 1

Plasmids are circular DNA molecules, relatively small, double stranded, extra-chromosomal, replicate independently of host chromosome

Question 2

What are non-recombinant plasmids?

Answer 2

Bacterial plasmids are double stranded closed circular DNA molecules (1 kB - 200 kB)

Behave as accessory genetic units that replicate and are inherited independently of the bacterial chromosome - self-replicating

Rely on enzymes and proteins encoded by the host for their replication and transcription (use their resources and machinery)

Question 3

What are the 5 steps for cloning plasmids/vectors?

Answer 3

1. Restriction endonuclease digest of DNA sample (cut DNA w/ restriction enzymes)
2. Restriction endonuclease digest of DNA plasmid/vector (digest circular molecules to become linear then add fragment)
3. Ligation of DNA sample fragments and vector (joins them together)
4. Transformation of competent cells (adding foreign plasmid to cells) with ligation
5. Growth on agar plates with selection for antibiotic resistance

Question 4

What is Type II restriction endonucleases?

Answer 4

Widely used in molecular cloning
Consist of a restriction endonuclease that cleave at a specific sequence (usually palindromic) of nucleotides in DS-DNA
Endonuclease - cleave in the middle
Exonuclease - cleave on the ends

Question 5

What is transformation?

Answer 5

Bacteria is treated with mixtures if divalent cations to make them temporarily permeable to small DNA molecules (make them competent)

Transformants are identified by selectable markers encoded by the plasmid used (markers confer a new phenotype - resistance to antibiotics)

Most common are ampicillin, tetracycline, etc.

Question 6

Why are smaller plasmids preferred

Answer 6

1. Efficiency of transformation is inversely related to the size of the plasmid
2. Smaller plasmids can accommodate larger fragments and still be efficiently transformed
3. Larger plasmids are more difficult to characterize by restriction mapping (more DNA, more restriction sites, harder to find and characterize)
4. Larger plasmids replicate to lower copy number, therefore yield of foreign DNA is reduced

Question 7

Plasmid replication

Answer 7

To replicate independently of the host cell chromosome a plasmid must have an origin of replication (ori)

A plasmid ori is a specific sequence of approx 300 nucleotides long which is required for replication

The seq is recognized by and bound by specific host proteins involved in initiation of DNA synthesis

The proteins open up (separate two strands) of DS-plasmid DNA forming a replication bubble

Change in conformation allows other host enzymes (DNA poly and primase) to enter each fork and carry out replication bidirectionally

Question 8

Multiple Cloning Site (MCS)

Answer 8

Plasmid vectors usually have a closely arranged series of restriction endonuclease sites called polylinker or MCS (all unique sites and only occur once in the plasmid)

Cutting the plasmid multiple times might lose parts of the plasmid

Question 9

Competent cells

Answer 9

Bacterial transformation are based on observations that bacteria treated with ice-cold solution of CaCl2 and then briefly heated (heat shock) can be transformed with plasmid DNA

The treatment induces a transient state of competence in the recipient bacteria (cells will be receptive to the foreign DNA)

Question 10

Procedure for CaCl2 Transformation

Answer 10

1. Centrifuge E.coli culture
2. Resuspend pellet in CaCl2 solution
3. Chill on ice, aliquot competent cells in microtubes
4. Add amp plasmid DNA into tubes, chill on ice
5. Heat shock in 42C water bath for 90 sec
6. Place on LB + ampicillin and incubate for 12-16 hours at 37C

Question 11

Why is CaCl2 used?

Answer 11

CaCl2 (Ca2+) binds to and interacts with negatively charged phospholipid and phosphates of DNA

Question 12

Transformation by high-voltage electroporation

Answer 12

Requires special machinery but is more efficient (can’t contain charges or it will fry the cells)

1. Centrifuge E.coli culture
2. Resuspend pellet in sterile H2O (NOT CaCl2)
3. Chill on ice, then centrifuge again
4. Resuspend pellet in sterile H2O (washing step)
5. Use competent cells immediately, add the plasmid DNA and electroshock in machinery
6. Add saline buffer and then plate on LB + amp plate

Question 13

Steps of Plasmid Isolation

Answer 13

1. Growth
2. Harvest Cells
3. Resuspend cells (GTE)
4. Lysis (NaOH/detergent)
5. Neutralization
6. Clearing the lysate
7. Collection of DNA
8. Resuspension (TE pH 8)

Question 14

How does number and size of plasmid affect the growth rate of the host?

Answer 14

The smaller the plasmid, the faster the growth rate

Cells with no plasmid grow the fastest because there is no competition with the host for replicative enzymes and metabolites (building blocks)