Isolasi Plasmid,

transformasi,
dan sekuensing

Umi Baroroh, S.Si.,

M.Biotek.
 Plasmids

Small circular DNA molecules that replicate independently of the

host chromosomes

Indispensable tools that allow molecular biologists to obtain

essentially unlimited amounts of a DNA sequence
 Plasmid
Circular and double-stranded

Plasmid size varies from 1 to over 100 kbp

The number of identical plasmids within a single

cell can be zero, one, or even hundreds under some
circumstances.
 • Fertility-F-plasmids, facilitate bacterial
conjugation

• Resistance-(R)plasmids, which
Plasmid contain genes that can build a
resistance against antibiotics or poisons
Classification • Col-plasmids, which contain genes that
based on its code for bacteriocins, protein that can kill
other bacteria
function • Degradative plasmids, which enable the
digestion of unusual substances, e.g.,
toluene or salicylic acid
• Virulence plasmids, which turn the
bacterium into a pathogen.
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.
https://www.youtube.com/watch?v=SdqJFA6mOkI
 We are using plasmids that have been termed shuttle vectors,
because they can be propagated in either bacteria or yeast

non-pathogenic strain Saccharomyces cerevisiae

of Escherichia coli deletion strains

Plasmids are propagated in Plasmid-encoded genes

bacteria, which grow quickly are expressed in yeast,
and maintain multiple copies and phenotypes are
of the plasmids analyzed

Shuttle vectors have origins of replication and selectable markers

for propagation in both bacteria and yeast
The pBG1805*-derived plasmids are complex vectors designed
to express S. cerevisiae ORFs

5 6 1 URA3

2 β-lactamase gene
3
ORF goes 3 pBR322 ori
here 1
2 4 yeast 2 µm origin

5 GAL1 promoter
4
6 C-terminal tags

*Plasmid names begin with a lower case “p”

 pYES2.1-based plasmids used for S. pombe ORFs have many
elements in common with pBG1805-based plasmids

1 URA3 3 pBR322 ori 5 GAL1 promoter

2 β-lactamase gene 4 yeast 2 µm origin 6 7 C-terminal tags

5 6 5
7

3
4
ORF goes
ORF goes 3
here 1
here
2

2
1
4

pBG1805 (6573 bp) pYES2.1 (5886 bp)

used for S. cerevisiae ORFs used for S. pombe ORFs
 Plasmid isolation is based on their distinctive physical
properties

Plasmids are much smaller than bacterial

chromosomes

Plasmids are supercoiled in their native form

Supercoiling allows plasmids to renature quickly

after they are denatured

Plasmids used in molecular biology are highly engineered and

contain elements of use to researchers
 • Miniprep
• Can be used to quickly find out whether the
Methods plasmid is correct in any of several bacterial
clones. The yield is a small amount of
to isolate impure plasmid DNA, which is sufficient for
analysis by restriction digest and for some
plasmid cloning techniques.
• Maxiprep/bulkprep
DNA • Much larger volumes of bacterial
suspension are grown from which a maxi-
prep can be performed. Essentially this is a
scaled-up miniprep followed by additional
purification. This results in relatively large
amounts (several micrograms) of very pure
plasmid DNA.
 Plasmid isolation from bacteria relies on their unique physical
properties

MANY different,
well-folded proteins

contains 1-2 copies of large

(>Mbp) , circular
bacterial DNA
Bacterial cell with
complexed with
plasmids
proteins

Multiple copies of small

(5-15 kbp) plasmids

Isolation involves sequential denaturation and renaturation steps

 Cells are first treated with base and a detergent

Proteins denature
irreversibly

Chromosomal DNA
denatures—will have
difficulty renaturing
because of its length
breaks open membrane and many proteins
and denatures both DNA complexed to it
and proteins

Plasmids denature, but

strands stay together
because of supercoiling
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 isolating plasmids, use a micropipette to remove the

supernatant for further processing steps
Plasmid DNA isolation
1. Inactivation of bacteria
2. Lysis of cells/ denaturation of DNA
3. Precipitation of DNA
4. Separate plasmid DNA from
contaminants
5. Precipitation of Plasmid DNA
6. Precipitation of proteins
7. Precipitate Plasmid DNA
Bacterial Transformation
• Transformation is the genetic alteration of a cell resulting from the
uptake and expression of foreign genetic material (DNA). i.e. the act
of putting foreign DNA into a bacterial cell

• Occurs in nature, but rarely

• If the foreign DNA has an origin of replication recognized by the host

cell DNA polymerases, the bacteria will replicate the foreign DNA
along with their own DNA.The origin of replication (also called the
replication origin) is a particular

sequence in a genome at which

replication is initiated
https://www.youtube.com/watch?v=w_fZmiIAdco
Bacterial Competence
• Not all bacteria are capable of taking up DNA from the
surrounding environment. Such bacteria are made artificially
competent. This is achieved by using chemicals and electrical
pulses.
• Chemicals- The cells are chilled and made permeable in the
presence of calcium phosphate. They are then incubated with
the DNA and provided with a heat shock treatment that causes
the DNA to enter the cells.
• Electroporation- The bacterial cells are subjected to electrical
pulses to make them permeable and cause the DNA to enter
into cells.
Atrificial competency
• SinceDNA is a very hydrophilic molecule, it won't normally
pass through a bacterial cell's membrane.
• Inorder to make bacteria take in the plasmid, they must
first be made "competent" to take up DNA.
• Thisis done by creating small holes in the bacterial cells by
different methods.
• Cells
that are undergoing very rapid growth are made
competent more easily than cells in other stages of growth.
1- Competency
• Label two sterile microtubes: one “+”and the other “-”.
• Using a disposable pipette, add 250 μl of 50mM CaCl2 solution
to each tube (“+” and “-”) and place them both on ice.
• Add 10 µl of ampicillin resistant plasmid
directly into the CaCl2 in “+” tube.
• Return the “+” tube to the ice.
DO NOT add the plasmid to your "-" tube. Incubate both tubes
on ice for 15 minutes.

Ca2+ neutralizes the negative charges

on the phosphate backbone of the DNA,
so that the DNA is not repulsed from the
phospholipids of the cell membrane.
This allows the DNA to more easily pass
1- Transformation
TRANSFORMATION EFFICIENCY
• After incubation count the colonies on each plate.
• Calculate the transformation efficiency
• Transformation Efficiency = no. of colonies observed/ µg
Plasmid
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