The document provides a comprehensive overview of plasmid DNA, including its fundamentals, applications, and laboratory techniques for manipulation. Key topics covered include plasmid replication methods, cloning strategies, plasmid purification techniques, and their uses in gene expression and transfer. Learning objectives emphasize understanding plasmid design and laboratory skills for extraction and analysis.

1. Plasmid DNA
Fundamentals and Laboratory Techniques

2. Contents
• Plasmid DNA: An overview
• Applications of Plasmid DNA
• Plasmid multiplication / cloning
• Plasmid extraction and purification

3. Learning Objectives
• To understand the fundamental aspects of plasmid
design.
• To develop the necessary laboratory skills for the
culture, maintenance extraction and manipulation of
plasmid DNA.
• To develop the necessary analytical skills required to
design plasmid vectors.

4. Learning Outcomes
• At the end of this lecture and associated practical session
(Cloning – II) you should be able to:
• 1. Define the conditions required for the culture of plasmids.
• 2. Extract and purify plasmid DNA.
• 3. Determine if your plasmid carries a foreign DNA fragment.
• 4. Design a functional plasmid based on what you have learnt.

5. What is a Plasmid?
• Extrachromosomal DNA.
• Replicates Independently of the Chromosome.
• Encode genes which do not have essential
functions.
• Genes which confer an advantage under specific
conditions.

6. What is a Plasmid?
• Size range: 2,000 bp to 100,000 bp.
• Negatively supercoiled.
• Copy number variable and determined by the type of
plasmid.
• Specific host range.
• Origin of replication (OriV)
• Plasmid designated with a lowercase ‘p’ as in pUC19

7. Plasmid Topology
• Supercoiled.
• Relaxed by nicking to give Circular topology.
• Linearized.

8. Some Naturally Occurring Plasmids
Plasmid Trait Source
pColE1 Bacteriocin E. coli
pTol Aromatic degradation P. putida
pTi Tumor initiation A. tumefaciens
pSym Nodulation R. meliloti
pRK2 Ampr
, Kanr
, Tetr
K. aerogenes

9. How do Plasmids Replicate?
•Theta Replication (Bidirectional).
•Rolling Circle Replication.

10. Theta Replication
• Plasmids rely on the host cell machinery to
replicate.
• DNA Polymerases.
• Ligases.
• Helicases.
• Primases.
• Replication initiated at the OriV

11. Rolling Circle Replication
• Rep Protein (Nicking Protein).
• DNA pol III (3’ -0H).
• DNA Ligase
• RNA Polymerase (Primase)
• DNA pol III (RNA Primed Replication)
• DNA pol I (Proofreading)

12. Relaxed & Stringent
• Relaxed: High Copy Number (300 – 1000)
• Stringent: Low Copy Number (1 – 20)

13. Feedback Regulation of Copy Number
In ColE1 plasmids, antisense RNA (RNA I) interferes with
the Primer (RNA II) and blocks replication by PolI.
The protein RopI stabilizes the interaction between RNA I
and RNA II and prevents it from priming the OriV.
The increase in the copy number of the plasmid results in
an increase in the number of RNA I transcripts which
subsequently regulate the copy number.
RNA I and RNA II are both encoded in the plasmid.

14. Copy Number
Copy number is regulated by the Origin of Replication.
Plasmid Origin of Replication Copy Number
pUC pMB1 500 - 700
pACYC p15A 10 - 12
pSC101 pSC101 5
Mutating the DNA sequence encoding the origin of replication
directly influences the copy number of plasmid DNA.

15. Shuttle Vectors
Shuttle Vectors are plasmids that can replicate in more than
one host, for example pKLAC1 can replicate in E. coli and
the yeast Kluyveromyces lactis.
https://www.neb.com/products/e1000-k-lactis-protein-expression-kit

16. What are the applications of Plasmid DNA?
• Genomic Library Construction.
• Heterologous Gene Expression.
• Third Generation Vaccines.
• Gene Delivery

17. Applications
• Genomic Library Construction.
• Concept of Double Digestion of Plasmid and
Genomic DNA.
• Plasmid copy number will be determined by the
size of the insert. Inverse relationship.

18. Cloning Vectors: Blunt
Blunt cloning vectors are ideal for cloning PCR products
generated using Proofreading DNA Polymerases.

19. Cloning Vectors: TA
TA Cloning Vectors are applied to clone PCR products
generated using non-proofreading DNA Polymerases.

20. Cloning Vectors: Overhang
Overhang Cloning Vectors are suitable for cloning DNA
which has been digested using two Restriction enzymes.

21. Applications: Gene Expression
• Heterologous Gene Expression involves the expression of
proteins in prokaryotic and eukaryotic hosts.
• Plasmid expression is driven by inducible promoters.
• The pET series of plasmids are the most commonly used
vectors for recombinant DNA expression.

23. Applications: Third Generation Vaccines
• Third Generation Vaccines are based on plasmid
DNA vectors which have been genetically
engineered to carry viral coat proteins under the
influence of a host specific promoter.
• Genetic material does not integrate into the
hosts genome.

25. Applications: Gene Transfer
• Gene Transfer into plants is done by using
plasmids based on the Ti plasmid derived from
the soil bacterium Agrobacterium tumefaciens.
• pCAMBIA is one of the most widely used plant
transformation vectors.
• Animal Cell lines: plasmid needs to be linearized
prior to transfection / transformation.

27. Methods in Plasmid Amplification
• Plasmid Amplification by cloning in bacterial hosts.
• Plasmid amplification by conventional PCR.
• Plasmid amplification by isothermal PCR using phi29 DNA
polymerase (High Fidelity RCA).

28. Plasmid Amplification: Cloning
Culture Conditions:
1. Temperature: low temperature will reduce plasmid copy
number but increase fidelity.
2. Media: supplementation with Phosphate ions will increase
plasmid copy number.

29. Plasmid Amplification: Cloning
Microbial Media used are:
1. Terrific Broth (Tryptone: 12 g, Yeast Extract: 24 g and
Glycerol: 4 ml dissolve to 900 ml in water, autoclave, after
autoclaving top up to 1000 ml with 0.17M KH2PO4 and
0.72M K2HPO4.
2. Lysogeny Broth (Tryptone: 10 g, Yeast Extract: 5 g and NaCl:
5g, dissolved in 1000 ml in water).

30. Plasmid Amplification: Conventional PCR
• Conventional PCR can be applied to amplify
plasmids by designing primers that anneal to the
terminal ends of the plasmid.
• Proofreading DNA Polymerase.
• Circularization using T4 DNA ligase.

31. Plasmid Amplification: Isothermal PCR
• Plasmid Amplification: Isothermal PCR.
• Plasmids can be amplified using DNA polymerase derived
from bacteriophage phi29.
• Primer used in Exo-resistant.
• Millions of copies of DNA

32. From Dean et al. 2001

33. Methods in Plasmid Purification
• Alkaline Lysis Miniprep
• Isopycnic Centrifugation.
• Solid Phase Extraction

34. Methods In Plasmid Purification: Alkaline Lysis
• Lysis of Bacterial Cells under alkaline conditions results in
the precipitation of genomic DNA and cellular proteins.
• Plasmid DNA which is relatively smaller that genomic
DNA remains in suspension.
• Can be used to separate most plasmids but has to be
modified in the case of plasmids with sizes in excess of
20,000 bp.

35. Methods In Plasmid Purification: Alkaline Lysis
• Solution I: Glucose (50 mM), EDTA.Na2 (10 mM) and Tris
HCl (25 mM) pH = 8.0. Supplements: Lysozyme and
RNase A.
• Solution II: NaOH (0.2 N) and SDS (1%).
• Solution III: Potassium Acetate (3M) pH = 4.8

36. Methods In Plasmid Purification: Solid Phase Extraction
• Solid phase extraction of Plasmids Involves Conventional
Alkaline Lysis Miniprep followed by binding to a
Chromatographic matrix.
• Lysozyme facilitates the disruption of the cell
membrane.
• The solutions used in SPE are Proprietary and generally
available in a kit format.

37. Methods in Plasmid Purification: Isopycnic

38. Impurities Associated with Plasmids
• Contamination with Lipo-polysaccharides which
are constituents of the bacterial cell wall.
• Collectively known as Endotoxins.
• Capable of eliciting an immune response when
delivered into organisms.

39. Assessment of Purity and Fidelity
• Gel Electrophoresis.
• Spectrophotometer.
• Restriction Analysis.
• PCR with MCS flanking primers.
• Sequencing.

40. Summary
• Characteristics.
• Replication
• Cloning of Plasmid DNA
• Purification of Plasmid DNA.