Plasmids Kinds Contributions Roles

1. Plasmids
Indispensable tools that allow molecular biologists to obtain
essentially unlimited amounts of a DNA sequence
Small circular DNA molecules that replicate independently of the
host chromosomes

2. Goals
• Learn about Plasmids,
• types, and
• mode of their replication.

3. Terminology
• Curing: process of eliminating plasmid from bacteria
• Episome: integration of plasmid with the chromosome

4. Bacterial Plasmids in Nature
1. Occur naturally in bacteria
and usually carry genes that
are useful but not essential
to survival: e.g. genes which
make bacteria resistant to
antibiotics.
2. Plasmids are released by
dead bacteria and absorbed
by those still living thus
genetic information is
exchanged (sexual
reproduction?).

5. Bacterial Plasmids in Nature
3. Some plasmids even
contain genes that
build a transfer tube
between bacteria.
4. There can be as many
as several hundred
copies of a single
plasmid in each
bacteria.

6. Plasmid Structure
1. Plasmids only need an “origin or
replication” and a “useful” gene
to be considered complete.
2. Molecular biologists have been
able to “insert” custom built
restriction sites into many
plasmids so they can be used to
“insert” DNA fragments from
other genes into them and thus
have a way to propagate those
DNA pieces.

7. Classification
On the basis of ability to perform conjugation:
Conjugative/self transmissible plasmid
Non conjugative plasmid
Based on compatibility b/w plasmid:
Compatible
Incompatible
Based on function:
Fertility/F plasmid: contain tra gene: sex pili expression
Resistance/R plasmid
Col plasmid
Virulence plasmid
Metabolic plasmid

8. • Usual double stranded closed cirle plasmids
• SS stranded circular DNA plasmids- Streptomyces, Clostridium
• Linear double stranded DNA plasmids in several bacteria and eukaryotic sources-Borrelia hermstii
• RNA plasimd – viroids are specialized in ss circular RNA plasmids carrying no genes
• Bipartite, linear ds RNA elements in yeast called killer factors
Based on structure

9. Classification
• Plasmids may be classified in a number of ways.
• Plasmids can be broadly classified into conjugative plasmids and non-conjugative plasmids.
Conjugative plasmids
• Conjugative plasmids contain a set of transfer or tra genes which promote sexual conjugation
between different cells.
• In the complex process of conjugation, plasmid may be transferred from one bacterium to
another via sex pili encoded by some of the tra genes.
Non-conjugative plasmids
• Non-conjugative plasmids are incapable of initiating conjugation, hence they can be
transferred only with the assistance of conjugative plasmids.
• An intermediate class of plasmids are mobilizable, and carry only a subset of the genes
required for transfer.
• They can parasitize a conjugative plasmid, transferring at high frequency only in its presence.

10. Incompatibility groups
• Plasmids can also be classified into incompatibility groups.
• A microbe can harbor different types of plasmids, however, different plasmids can only exist in a single
bacterial cell if they are compatible.
• If two plasmids are not compatible, one or the other will be rapidly lost from the cell.
• Different plasmids may therefore be assigned to different incompatibility groups depending on whether
they can coexist together.
• Incompatible plasmids normally share the same replication or partition mechanisms and can thus not be
kept together in a single cell.
• Plasmids that are able to coexist in the same cell do not interfere with each other’s replication

11. Plasmid Biology
1. Plasmid structure
2. Plasmid replication and copy number control
3. Plasmid transfer
4. Plasmids as tools
5. F plasmids

12. 1. Extrachromosomal DNA, usually circular-parasite?
2. Usually encode ancillary functions for in vitro growth
3. Can be essential for specific environments: virulence, antibiotics resistance, use of unusual nutrients, production of
bacteriocins (colicins)
4. Must be a replicon - self-replicating genetic unit
5. Plasmid DNA must replicate every time host cell divides or it will be lost
a. DNA replication
b. partitioning (making sure each progeny cells receives a plasmid)
6. High copy plasmids are usually small; low copy plasmids can be large
7. Partitioning is strictly controlled for low copy, but loose for high copy
8. Plasmid replication requires host cell functions
9. Copy number is regulated by initiation of plasmid replication
10. Plasmids are incompatible when they cannot be stably maintained in the same cell because they interfere with
each other’s replication.
Functions

13. Also, virulence plasmids from
Salmonella, Shigella, Yersinia,
B. anthracis, E.coli, and others.

14. Based on phenotypes
Enterotoxin production - Enterobacteria Ent P 307
Heavy metal resistance - Pseudomonas sps FP2
Tumor induction - Agrobacterium tumefaciens
U.V. protection - plasmid R46
Senescence - Neurospora crassa
Bacteriocin production , HS production , degradation of aromatic compounds
Phage Resistance
Sugar fermentations

16. Plasmid replication
1. Plasmid replication requires host DNA replication machinery.
2. Most wild plasmids carry genes needed for transfer and copy number control.
3. All self replication plasmids have a oriV: origin of replication
4. Some plasmids carry and oriT: origin of transfer. These plasmids will also carry functions needed
to be mobilized or mob genes.
5. Plasmid segregation is maintained by a par locus-a partition locus that ensures each daughter cells
gets on plasmid. Not all plasmids have such sequences.
6. There are 5 main “incompatibility” groups of plasmid replication. Not all plasmids can live with
each other.
7. Agents that disrupt DNA replication destabilize or cure plasmids from cells.

17. Types of plasmid replications
• Well-known replication systems of circular plasmids include
a) Theta-type replication,
b) Rolling-circle replication, and
c) Strand displacement-type replication

18. Theta type replication
• Theta mode of replication is similar to chromosomal replication.
• There is the synthesis of leading- and lagging-strand.
• Lagging-strand is discontinuous.
• No DNA breaks are required for this mode of replication.
• There is the formation of bubbles in the early stages of replication.
• It resembles the Greek letter θ.
• Theta replication is of 4 types:
– θ class A
– θ class B
– θ class C
– θ class D

19. Rolling circle model
• Rolling circle replication mechanism is specific to bacteriophage
family M13 and the fertility F factor which encodes for sex pili
formation during recombination by means of conjugation.
• Fragments smaller than 10 kilo base (Kb) usually replicate by this
replication mechanism as reported in some gram positive bacteria.
• It allows the transfer of single stranded replication product at a
faster rate to the recipient cell through pilus as in case of fertility
factor or to the membrane in case of phage
• Rolling circle occurs to a covalently closed circular piece of
double-stranded DNA.
• A nick is produced in one of the strands by enzyme nickases
creating a 5’ phosphate and a 3’ hydroxyl.
• Free 3’ hydroxyl will be used by DNA polymerase to make new
DNA pushing the old nicked strand off of the template DNA

20. Single strand displacement model
• Replication begins at one end of linear genome (=
chromosome), eg: in adenovirus
• Only one of the two strands i.e. the strand oriented in 3’ to
5’ direction of the DNA duplex is replicated in a
continuous manner.
• This produces a DNA duplex and a single strand (the 5’ to
3’ strand).
• The single strand base pairs at its ends to form a circle:
replication of this strand now proceeds in a continuous
manner
• In adenovirus, a protein attached to the 5’ end provides the
primer function
• This protein has a cytosine residue attached to it: the free
3’-OH of this C is used as primer.

21. Site-directed mutation: Suicide plasmds
1. Plasmid must be unable to replicate without essential replication proteins provide in trans.
2. It helps if the plasmid can be mobilized-oriT required
3. Need a selectable marker
4. Large or small region of homologous DNA cloned that will integrate into the chromosomal
target.
5. Need a counter selection method to kill the donor cells
6. Screen for what you think is correct.
Also, merodiploid reporter strains can be constructed in this manner
1. Make a lacZ fusion to your promoter of interest
2. Clone into a suicide plasmid
3. Mate into recipient.
4. Resulting strain will harbor a duplication of the promoter region:lacZ and still have a functional
copy of the gene.
Why would this be important?
Plasmids as genetic tools: Construction of Mutants

22. Plasmids can be manipulated easily in the laboratory
1. Plasmids can be collected from bacteria.
2. Restriction enzymes can be purified and used to identify and cut out specific sequences of DNA
along with the plasmid vector.
3. Ligase (enzyme) can be purified and used to “glue” pieces of DNA together.
4. Bacteria can be transformed by taking in plasmids given to them.

23. Transforming Bacteria
• When a bacterial cell takes in a plasmid
from the environment, it has new DNA
(and therefore, new traits).
• Scientists say that the bacteria has been
transformed.

24. How to produce human insulin from bacteria and
become a multimillionaire
1. Isolate plasmid DNA from bacteria and
insulin gene from human.
2. Cut both DNAs with the same restriction
enzyme.
3. Mix the DNA together with ligase.
4. Insert the new DNA into bacteria
(transformation).
5. Use antibiotics to kill any bacteria without
the plasmid.
6. Grow bacteria and harvest the insulin.
Insulin
Purification
Insulin gene
Human DNA

25. Transfer of plasmids can occur by four routes
1. Cell fusion
2. Transformation
3. Transduction
4. Conjugation

26. Conjugative plasmids

27. Hfr X F- crosses

28. This mechanism explains the characteristics of
F' X F- crosses. The F- becomes F', the F'
remains F' and the is high frequency transfer of
donor genes on the F' but low frequency
transfer of other donor chromosomal genes.

29. 1. large (100 kb)
2. low copy (1-2 copies/cell)
3. self transmissible
4. requires protein synthesis (chloramphenicol-sensitive)
5. repE gene encodes RepE protein
6. RepE protein binds to origin of replication (oriS) and initiates
DNA replication
7. RepE binds to the repE promoter and activates transcription
8. RepE binds to the copA/incC locus binding copies of F
together via RepE – inhibiting replication (coupling)
F-plasmid

30. F-transfer at fine detail

31. How are plasmids constructed?
What functional elements are found in our yeast
overexpression plasmids?
How are plasmids purified?

32. Plasmids used in molecular biology have been constructed in the lab
Molecular cloning
Enzymes are used to insert desired
pieces of foreign DNA into plasmids
Bacterial cells are transformed with
the plasmids. Copies of the
plasmids are purified from bacteria.

33. Shuttle vectors have origins of replication and selectable markers
for propagation in both bacteria and yeast
We are using plasmids that have been termed shuttle
vectors, because they can be propagated in either bacteria
or yeast
Plasmids are propagated in
bacteria, which grow quickly
and maintain multiple copies
of the plasmids
non-pathogenic strain
of Escherichia coli
Saccharomyces cerevisiae
deletion strains
Plasmid-encoded genes
are expressed in yeast,
and phenotypes are
analyzed

34. How are plasmids constructed?
What functional elements are found in our yeast
overexpression plasmids?
How are plasmids purified?

35. How are plasmids constructed?
What functional elements are found in our yeast
overexpression plasmids?
How are plasmids purified?

36. Plasmids are much smaller than bacterial
chromosomes
Plasmids are supercoiled in their native form
Supercoiling allows plasmids to renature quickly
after they are denatured
Plasmid purification is based on their distinctive
physical properties
Plasmids used in molecular biology are highly engineered and
contain elements of use to researchers

37. Plasmid purification from bacteria relies on their unique
physical properties
Bacterial cell with
plasmids
contains
MANY different,
well-folded proteins
1-2 copies of large
(>Mbp) , circular
bacterial DNA
complexed with
proteins
Multiple copies of small
(5-15 kbp) plasmids
Purification involves sequential denaturation and renaturation steps

38. Cells are first treated with base and a detergent
breaks open membrane
and denatures both DNA
and proteins
Proteins denature
irreversibly
Chromosomal DNA
denatures—will have
difficulty renaturing
because of its length
and many proteins
complexed to it
Plasmids denature, but
strands stay together
because of supercoiling

39. Extract is neutralized to allow DNA molecules to renature
Plasmids
renature and are
suspended in the
SUPERNATANT
following
centrifugation
Proteins and
chromosomal DNA form
aggregate irreversibly,
forming a PRECIPITATE
that can be collected by
centrifugation
When purifying plasmids, use a micropipette to remove the
supernatant for further processing steps

40. Isolation and purification of Plasmid
MiniPrep procedure

41. PLASMID ISOLATION AND ANALYSIS: Part II
Pellet 2 ml of overnight cell suspension by adding 1 ml (20 drops)
of overnight culture suspension into a 1.5 ml reaction tube.
Spin for 1 minute at 10,000 rpm in a microcentrifuge (MicroV).
Plasmid Purification and Isolation
Step 1

42. PLASMID ISOLATION AND ANALYSIS: Part II
A pellet should form in the bottom of the tube. Drain off the
liquid into a designated container and gently tap the tube on a
paper towel to remove the excess broth. Repeat the procedure
with a second 1 ml volume of overnight culture suspension.
Plasmid Purification and Isolation
Step 1 (cont.)

43. PLASMID ISOLATION AND ANALYSIS: Part II
Use a small needle pipette to add the contents of the tube marked
“Cell Lysis Solution”(P2) to the Resuspension tube and mix by
inverting the tube 4 times. The cell suspension should appear clear.
Plasmid Purification and Isolation
Step 3

44. PLASMID ISOLATION AND ANALYSIS: Part II
Use a small needle pipette to add the contents of the tube marked
“Neutralization Solution” (N3) to the Resuspension tube and mix
by inverting the tube 4 times.
Plasmid Purification and Isolation
Step 4

45. PLASMID ISOLATION AND ANALYSIS: Part II
Centrifuge the tube containing the lysate at 10,000 rpm for 10 minutes.
Plasmid Purification and Isolation
Step 5

46. PLASMID ISOLATION AND ANALYSIS: Part II
While the lysate is in the centrifuge, label two 1.5 ml tubes.
Label the lid and side of one tube “L”.
Plasmid Purification and Isolation
Step 6

47. PLASMID ISOLATION AND ANALYSIS: Part II
Label the lid and side of the second tube “M.” Remove the lid
from the tube with a scissors. Place the lid back on the tube and
hold until Part III, step 7.
Plasmid Purification and Isolation
Step 7

48. PLASMID ISOLATION AND ANALYSIS: Part II
Use the small needle pipette to carefully remove all of the clear
lysate/supernate from the miniprep in step 5. Do not remove any of
the precipitate. Wipe the end of the pipette with a chem wipe or
kleenex to remove any protein/chromosomal DNA precipitate.
Plasmid Purification and Isolation
Step 8

49. PLASMID ISOLATION AND ANALYSIS: Part II
Pipette the clear lysate/supernate into the tube marked “L.”
At this point you can choose to store the tube in the refrigerator
until the next day or, if time permits, continue to Part III.
Plasmid Purification and Isolation
Step 9

50. Zyppy purification kit use multiple steps to purify plasmids
Alkaline lysis
Neutralization
Purification of plasmid DNA on a silica resin
Elution of purified DNA from he silica resin
Let's look at the individual steps……………..

51. CsCl gradient with
ethidium bromide and
UV light.
Three forms of plasmid DNA
“Old School method of purifying plasmid”

52. 1 Transformed E. coli cultures are
concentrated by centrifugation
2. The cell pellet is resuspended
in 600 µL TE buffer by vortexing
3. 100 µL of 7X Blue Zyppy lysis buffer
is added
0.1 N NaOH in buffer lyses the cells
GENTLY mix the contents by inverting the tube 4-6 times
Solution changes from cloudy to clear when cells are lysed
Warning: too much mechanical agitation can shear chromosomal DNA
Alkaline lysis

53. Neutralization
4. Add 350 µL yellow Zyppy
Neutralization buffer
Mix by inverting several times
A heavy precipitate will begin to form immediately!
The initial “glop” will become more granular when
neutralization is complete—but don’t overdo it!
The precipitate contains denatured proteins and
the denatured chromosomal DNA.
5. Spin down the denatured molecules
for 3 minutes at top speed. CAREFULLY
remove the supernatant containing the
plasmid – Don't be greedy! Purity is
preferred to yield!

54. Purification on
Zyppy silica resin
6. Apply the supernatant to the spin
column. Place the column in the
collection tube. Centrifuge the column
for ~15 seconds at top speed.
7. Discard the flow through in the
collection tube. Add 200 µL Zyppy
EndoWash. Centrifuge ~15 sec.
EndoWash contains guanidine
hydrochloride and
isopropanol. It removes
contaminating proteins that
are bound to the resin.
8. Discard the flow through in the
collection tube. Add 400 µL Column
Wash. Centrifuge 1 min.

55. Plasmid Elution
9. Transfer the column to a clean,
LABELED microcentrifuge tube
10. Add 50 µL TE buffer directly on
top of the column. Allow the column to
stand upright in the test tube for ~10
min. (Plasmid is being eluted.)
11. Spin the column for 30 seconds.
Plasmid DNA will be collected in the
microcentrifuge tube. Pure plasmid DNA
collects here!