A plasmid is a small, often

circular DNA molecule found

in bacteria and other cells.
Plasmids are separate from
the bacterial chromosome
and replicate independently
of it. They generally carry
only a small number of
genes, notably some
associated with antibiotic
resistance. Plasmids may be
passed between different
bacterial cells.
Each bacterial cell typically produces many copies of a plasmid, in contrast to making only one copy of its
own chromosome.
The fact that plasmids are smaller and in greater number than the host chromosome make plasmids easier
to isolate in pure form, which is why they are commonly in use them DNA in the laboratory.
Plasmids are thus a fundamental tool of recombinant DNA technology.
Recombinant DNA technology enables individual fragments of DNA to be inserted into vector DNA
molecules, such as plasmids, and amplified in bacteria. Each amplified fragment is called a DNA clone.
The breakthrough that made
recombinant DNA technology possible
was the discovery and The enzyme EcoRI cuts within this
characterization of restriction sequence but in a pair of
enzymes. staggered cuts between the G and
As an example: the restriction the A nucleotides.
enzyme EcoRI (from E. coli) This staggered cut leaves a pair of
recognizes the following six- identical single-stranded “sticky
nucleotide-pair sequence in the DNA ends.” The ends are called sticky
of any organism: because they can hydrogen bond
(stick) to a complementary
sequence.

This type of segment is called a DNA

palindrome, which means that both
strands have the same nucleotide
sequence but in antiparallel
orientation. Many different restriction
enzymes recognize and cut specific
palindromes.
EcoRI makes a single cut in a circular
DNA molecule such as a plasmid: the
cut opens up the circle, and the linear
molecule formed has two sticky ends.
Production of these sticky ends is a
feature of restriction enzymes that
makes them suitable for recombinant
DNA technology. The principle is
simply that, if two different DNA
molecules are cut with the same
restriction enzyme, both will produce
fragments with the same
complementary sticky ends, making it
possible for DNA chimeras to form.
Hence, if both vector DNA and donor
DNA are cut with EcoRI, the sticky
ends of the vector can bond to the
sticky ends of a donor fragment when
the two are mixed.
The two DNA
backbones can be
sealed by the addition
of the enzyme DNA
ligase, which create
phosphodiester bonds
at the junctions.

The ligated recombinant DNA enters
a bacterial cell by transformation.
After it is in the host cell, the plasmid
vector is able to replicate because
plasmids normally have a replication
origin. However, now that the donor
DNA insert is part of the vector’s
length, the donor DNA
automatically replicated along with
the vector.
Each recombinant plasmid that
enters a cell will form multiple copies
of itself in that cell.
The introduction of DNA into
bacteria by transformation is an
essential step in the construction of
recombinant strains. Electroporation
(electropermeabilization), in which a
brief high voltage electric discharge
is used to render cells permeable to
DNA.
is
Otherwise, by exposing cells to a
sudden increase in temperature, or
heat shock, a pressure difference
between the outside and the inside
of the cell is created, that induces
the formation of pores, through
which supercoiled plasmid DNA can
enter.
Subsequently, many cycles of cell
division will take place, and the
recombinant vectors will undergo
more rounds of replication. The
resulting colony of bacteria will
contain billions of copies of the
single donor DNA insert. This set
of amplified copies of the single
donor DNA fragment is a DNA
clone.
After transformation, bacteria
are selected on antibiotic plates.
Bacteria with a plasmid are
antibiotic-resistant, and each
one will form a colony. Colonies
with the right plasmid can be
grown to make large cultures of
identical bacteria.
Each spot in the picture is a
bacterial clone: A large
colony of identical bacteria
with engineered plasmids
harboring the right antibiotic
resistance gene.
BioBrick standard assembly 10: the general scheme (Tom Knight, 2003)
isocaudamers

‘scar’

Idempotent vector design for standard assembly of BioBricks: the general scheme
? A standard gene layout
 =
‘scar’

Examples of BioBricks
in the Registry
Step 1

then

A possible scheme to
assemble a gene
circuit

Step 2
The assembled gene
then circuit = another
BioBrick in the Registry

Step 3
Examples of BioBricks
in the Registry
Step 1a Step 1b

then
Another possible, and
faster, scheme to
assemble a gene circuit

Step 2

The assembled gene

circuit = another
BioBrick in the Registry
Each single step is a molecular cloning operation.

It takes about one day to be completed.

Day 1 - a

EcoRI
A B
Cutting BioBrick 1 with Cutting BioBrick 2 with
E & S - ~30’ (@ 37 °C) E & X - ~30’ (@ 37 °C) SpeI

XbaI

Run the samples in agarose gel electrophoresis – ~90’

Day 1 - b

Purify and ligate DNA – total procedure ~90’

Cut the fragments of Purify DNA (each single Ligate the fragments
interest out of the gel fragment separately)
Day 1 - c

Bacterial transformation – total procedure ~90’

plasmid

Transformation of plasmid into

bacterial cells* – ~90’

*NB: the aim is to increase the

amount of engineered DNA

E. Coli

NB: You may like to browse https://www.addgene.org/protocols/subcloning/

Day 1 - d

Bacterial plating and grow of colonies – total procedure overnight’

E. Coli +
Plate transformed bacterial cells over plasmid(s)
LB Agar * – ~5’

Let bacteria grow in antibiotic-

resistant colonies *, each of them = a
single clone – overnight (@ 37 °C)
E. Coli
*NB: the aim is to increase the colonies in
amount of engineered DNA LB agar in a
Petri dish

Notice
Day 2 - a

Bacterial inoculation into a liquid culture – total procedure ~6-8 hrs

Pick a clone a let it expand* into LB

liquid medium – ~6-8 hrs (@ 37 °C)

*NB: this step further increase the

amount of engineered DNA
Day 2 - a

Plasmid DNA purification – total procedure ~60’

Harvest and homogenize

bacteria, purify plasmid =
DNA – ~60’

NB: It takes about two days working to get a single molecular cloning procedure finished !
 =
‘scar’

Step 1

then

A full week of work

is needed to
assemble the entire
gene circuit! Step 2
The assembled gene
then circuit = another
BioBrick in the Registry

Step 3