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Lecture 2

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Lecture 2

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maittt.22ba13211
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Lecture 2

After this lecture, students can:


- Analyze the difference between genome of eukaryote and prokaryote

- Describe main components of plasmid, types of restriction enzymes and its


function in the laboratory
Genome organization

☑ Prokaryotes
-Most genome is coding, no introns
-Small amount of noncoding is regulatory sequences
☑ Eukaryotes
-Most genome is noncoding
-Regulatory sequences
-Intron
-Repetitive DNA
Introns
● The human genome has about ten times the DNA content
as that of yeast.

● However, humans do not have a substantially larger


number of coding genes than yeast: humans genes are more
spread out over more chromosomes, and typically have
a higher proportion of introns.
Chromosome and plasmid

Plasmid DNA

Bacterial Chromosomal DNA

Plasmid in prokaryote cells

Plasmid also occur in some eukaryotes. Which Eukaryote?


Plasmid in the nucleus of most Saccharomyces cerevisiae strains

Plasmid in the chloroplasts of Chlamydomonas reinhardtii.


Plasmid

● A plasmid is a small, circular, double-stranded DNA molecule that is


distinct from a cell's chromosomal DNA.
● Plasmids naturally exist in bacterial cells, and some eukaryotes.
● Genes carried in plasmids provide bacteria with genetic advantages,
such as antibiotic resistance.
● Plasmids have a wide range of lengths, from roughly one thousand
DNA bps to hundreds of thousands of bps.
● When a bacterium divides, all of the plasmids are copied and
transmitted to daughter
● Bacteria can also transfer plasmids to one another through
conjugation.
Plasmid in the lab

● As tools to clone, transfer, and manipulate genes: vectors.

DNA fragments or genes can be inserted into a plasmid vector,


creating a recombinant plasmid.

● This plasmid can be introduced into a bacterium by


transformation.

● Then, because bacteria divide rapidly, they can be used as factories


to copy DNA fragments in large quantities.
A plasmid is a circular dsDNA molecule a few
hundred or thousand base pairs in circumference.

Naturally-occurring plasmids are viruses of bacteria.

The artificial plasmid pUC18 has been genetically


engineered to include (1) a gene for antibiotic
resistance to Ampicillin (ampR), and (2) a gene (and
its promoter) for the enzyme beta-
galactosidase (lacZ).

The lacZ gene contains a (3) polylinker region, with


a series of unique restriction sites found nowhere
else in the plasmid.

Digestion with any one of these endonucleases will


make a single cut that linearizes the circular
plasmid DNA, and allow it to recombine with
foreign DNA that has been cut with the same
endonuclease.
Regulatory sequences (promoter)
● CAAT box. A consensus sequence close to -80 bp from the start
point (+1). It plays an important role in promoter efficiency
This box is replaced in plants by a consensus sequence called the
AGGA box;
CAAT not found in prokaryotes.

● TATA box. A sequence usually located around 25 bp upstream of the


start point. The TATA box tends to be surrounded by GC rich
sequences. The TATA box binds RNA polymerase II and a series of
transcription factors (TFIIX, X being a letter that identifies an
individual transcription factor) to form an initiation complex

● GC box. A sequence rich in GC nucleotides, is usually found in


multiple copies in the promoter region, normally surrounding the
TATA box.
Multiple Cloning Region

Multiple
Cloning
Region

The cloning marker for this plasmid is the lacZ gene.


Restriction enzymes
 Are DNA-cutting enzymes, are found in bacteria (and other prokaryotes). They recognize
and bind to specific sequences of DNA, called restriction sites.

 4 to 8 base pairs (bp) in length

 Each enzyme recognizes one or a few target sequences and cuts DNA at or near those
sequences.

 Producing ends with single-stranded DNA overhangs. However, some produce blunt
ends.

 DNA ligase is a DNA-joining enzyme. If two pieces of DNA have matching ends, ligase
can link them to form a single, unbroken molecule of DNA.

 In DNA cloning, restriction enzymes and DNA ligase are used to insert genes and other
pieces of DNA into plasmids.
Transforming Bacteria

 After you create your new plasmid construct that contains your insert
of interest , you will need to insert it into a bacterial host cell so that it
can be replicated.

 The process of introducing the foreign DNA into the bacterial cell is
called transformation.
Competent Host Cells

 Not every bacterial cell is able to take up plasmid DNA.

 Bacterial cells that can take up DNA from the environment are said to be competent. (at
which stages?)

 Can treat cells (electrical current/divalent cations salts) to increase the likelihood that
DNA will be taken up

 Two methods for transforming: heat shock and electroporation

Video
Selecting for Transformants
 The transformed bacteria cells are grown on selective media (containing
antibiotic) to select for cells that took up plasmid.

 For blue/white selection to determine if the plasmid contains an insert, the


transformants are grown on plates containing X-Gal and IPTG.
lacZ gene (codes for beta-galactosidase)
23

BamH1
ApR beta-galactosidase
lacZ cleaves X-gal and
ori
produces a blue color
O
O Cl OH
O Br HO Cl Br
X-gal N
(CLEAR) beta-galactosidase N BLUE
product
Blue-white selection
Colonies containing vector WITHOUT an insert are blue.
25

BamH1
ApR
lacZ
+ ampicillin
ori
+ X-gal

(We don't want these.)


When foreign DNA is inserted, it inactivates lacZ
26

beta-galactosidase is not made


X-gal is not cleaved
colonies with insert are white, NOT blue

insert
X (LacZ-)

X-gal
(CLEAR) X + ampicillin
+ X-gal
Selectable markers and gene inactivation
27 BamH1 Uncut vector allows cells to grow on ampicillin
ApR (Ap) and tetracycline (Tc).
TcR

ori
transform
E. coli
replica
plating + ampicillin
+ ampicillin
+ tetracycline

What happens when foreign DNA is inserted


into the BamH1 site?

• the TcR (tetracyline resistance) gene is inactivated


Selectable markers and gene inactivation
28 BamH1
ApR
TcR
ApR
ori ApR TcR
TcR
BamH1
digest insert
+

ApR

When foreign DNA is inserted,


• TcR gene is inactivated
• cells will grow on Ap, but NOT tetracycline
In this gene inactivation system, what happens when E. coli is
transformed with a mixture of vector and [vector with insert]?
29

replica
plating + ampicillin
+ ampicillin
+ tetracycline

Cells containing the cloned DNA (insert), are Ap-resistant (ApR) but Tc-
sensitive (TcS ).
Fluorescent gene insertion
Concept of transgenic technology

2008 Nobel Prize in Chemistry awarded to Shimomura, Chalfie, and Tsien


“for the discovery and development of the green fluorescent protein, GFP”
Variation of fluorescence
proteins
How fluorescence protein works?
How fluorescence protein works?
Fluorescent gene insertion
 A plasmid is a small DNA molecule, is physically
separated from a chromosomal DNA and can
replicate independently.

 They are most commonly found as small circular,


double-stranded DNA molecules in bacteria;  Artificial plasmids are widely used
sometimes present in archaea and eukaryotic as vectors in molecular cloning, serving to drive
organisms. the replication of recombinant DNA sequences
within host organisms.
 In nature, plasmids often carry genes benefit for
survival of the organism under certain  In the laboratory, plasmids may be introduced into
situations, for example antibiotic resistance. a cell via transformation.
Plasmids enter the cell easily by nature or
in the lab

Account for rapid spread of antibiotic resistant genes

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