Gene cloning, expression

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Gene cloning, expression and functional
study ????,???????
2
vectors
Cloning vectors ???? to clone a gene in a
vector Expression vectors ???? to express a
gene from a vector Integration vectors ???? to
integrate a gene in a genome through a vector
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Cloning vectors
1 Plasmid vecters 2 Bacteriophage vectors 3
Cosmids BACs 4 Eukaryotic vectors
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Cloning vectors allowing the exogenous DNA to be
inserted, stored, and manipulated mainly at DNA
level.
expression vectors allowing the exogenous DNA to
be inserted, stored, and expressed.
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A plasmid vector for cloning

• Contains an origin of replication, allowing for
  replication independent of hosts genome.
• Contains Selective markers Selection of cells
  containing a plasmid
• twin antibiotic resistance
• blue-white screening
• Contains a multiple cloning site (MCS)
• Easy to be isolated from the host cell.

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Twin antibiotic resistance screening
-Screening by insertional inactivation of a
resistance gene
B X B
B
Tcr
Ampr
Ampr
X
B
ori
ori
Ampicillin resistant? yes
yes Tetracycline resistant? No
yes
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Replica plating transfer of the colonies from
one plate to another using absorbent pad or
Velvet (??).
transfer of colonies
ampicillin tetracycline
ampicillin
these colonies have bacteria with recombinant
plasmid
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Blue white screening
Screening by insertional inactivation of the lacZ
gene
Lac promoter
MCS (Multiple cloning sites, ?????)
Ampr
pUC18 (3 kb)
lacZ
ori
The insertion of a DNA fragment interrupts the
ORF of lacZ gene, resulting in non-functional
gene product that can not digest its substrate
x-gal.
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Recreated vector blue transformants Recombinant
plasmid containing inserted DNA white
transformants
Recreated vector (no insert)
Recombinant plasmid (contain insert)
back
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Multiple cloning sites
Multiple restriction sites enable the convenient
insertion of target DNA into a vector
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A plasmid vector for gene expression
Expression vectors allowing the exogenous DNA to
be inserted, stored and expressed.

• Promoter and terminator for RNA transcription are
  required.
• Intact ORF and ribosomal binding sites (RBS) are
  required for translation.
• Include(1) bacterial expression vectors, (2)
  yeast expression vectors, (3) mammalian
  expression vector

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T7 promoter
RBS
Start codon
MCS
Ampr
Transcription terminator
T7 expression vector
ori
An bacterial expression vector
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A yeast expression vector
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Bacteriophage vector

• Two examples
• ? phage
• bacteriophage?
• ? replacement vector
• M13 phage
• M13 phage vector
• Cloning in M13
• Hybrid plasmid-M13 vectors

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? phage

• viruses that can infect bacteria.
• 48.5 kb in length
• Linear or circular genome (cos ends)
• Lytic phase (Replicate and release)
• Lysogenic phase (integrate into host genome)

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Cloning large DNA fragments
(Eukaryotic Genome project)
Analysis of eukaryotic genes and the genome
organization of eukaryotes requires vectors with
a larger capacity for cloned DNA than plasmids or
phage ?. Human genome (3 x 109 bp) large genome
and large gene demand vectors with a large size
capacity.
Genomic library VS cDNA library
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Cosmid vectors

• Utilizing the properties of the phage l cos sites
  in a plasmid vector.
• A combination of the plasmid vector and the COS
  site which allows the target DNA to be inserted
  into the l head.
• The insert can be 37-52 kb

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Formation of a cosmid clone
C) Packaging and infect
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YAC vectors
(yeast artificial chromosome)
Accommodates genomic DNA fragments of more than 1
Mb, and can be used to clone the entire human
genome, but not good in mapping and analysis.
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• Essential components of YAC vectors
• Centromers (CEN), telomeres (TEL) and
  autonomous replicating sequence (ARS) for
  proliferation in the host cell.
• ampr for selective amplification and markers
  such as TRP1 and URA3 for identifying cells
  containing the YAC vector in yeast cells.
• Recognition sites of restriction enzymes (e.g.,
  EcoRI and BamHI)

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YAC Cloning
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BAC vectors ??????? 1. More stable than YAC 2.
Capacity is 300-350 kb 3. One to two copies in
each cell 4. Easy to handle 5. More popular in
genomic mapping
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I1 Genomic libraries
Gene libraries and screening

• I1-1 Representative gene libraries
• I1-2 Size of library
• I1-3 Genomic DNA
• I1-4 Vectors

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I1 Genomic libraries
Gene library a collection of different DNA
sequence from an organism, each of which has been
cloned into a vector for ease of purification,
storage and analysis.
Genomic libraries
(made from genomic DNA)
Gene library
cDNA libraries
(made from cDNA- copy of mRNA)
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I1 Genomic libraries
I1-1 Representative gene libraries
--- Contain all the original sequences
Missing original sequence

• Certain sequences have not been cloned.
• Example repetitive sequences lacking
  restriction sites

Too long for the vector used
2. Library does not contain sufficient clones
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I1 Genomic libraries
I1-2 Size of library (ensure enough clones)
must contain a certain number of recombinants
for there to be a high probability of it
containing any particular sequence
The formula to calculate the number of
recombinants
ln (1-P)
N
ln (1-f)
P desired probability f the fraction of
the genome in one insert
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I1 Genomic libraries
For example for a probability of 0.99 with
insert sizes of 20 kb these values for the
E.coli (4.6106 bp) and human (3109 bp)
genomes are N E.coli
1.1 103
ln( 1-0.99)
ln1-(2104/4.6106)
ln(1-0.99)
Nhuman
6.9 105
ln1-(2 104/3 109)
These values explain why it is possible to make
good genomic libraries from prokaryotes in
plasmids where the insert size is 5-10kb ,as
only a few thousand recombinants will be
needed.
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I1-3 Genomic DNA libraries
I1 Genomic libraries
eukaryotes

• Purify genomic DNA
• Fragment this DNA physical shearing and
  restriction enzyme digestion

prokaryotes
Clone the fragments into vectors
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I1 Genomic libraries
To make a representative genomic libraries
, genomic DNA must be purified and then broken
randomly into fragments that are correct in size
for cloning into the chosen vector.
Purification of genomic DNA
Eukaryotes prepare cell nuclei

remove protein, lipids and other unwanted
macro- molecules by protease digestion and phase
extraction.
Prokaryotes extracted DNA directly from cells
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I1 Genomic libraries
Break DNA into fragments randomly
Physical shearing pipeting, mixing or
sonicaion

Restriction enzyme digestion partial
digestion is preferred to get a greater lengths
of DNA fragments.
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I1 Genomic libraries
Selection of restriction enzyme

• Ends produced (sticky or blunt)
• The cleaved ends of the vector to be cloned

Sau3A 5-/GATC-3, less selectivity BamH1
5-G/GATCC

• Whether the enzyme is inhibited by DNA
  modifications (CpG methylation in mammals
• Time of digestion and ratio of restriction enzyme
  to DNA is dependent on the desired insert size
  range.

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I1 Genomic libraries
I1-4 Vectors
According to genomes size,we can select a proper
vector to construct a library .
Vectors Plasmid phage? cosmid
YAC insert (kb) 5 23
45 1000
The most commonly chosen genomic cloning vectors
are ? relacement vectors which must be digested
with restriction enzymes to produce the two ?
end fragment or ? arms between which the genomic
DNA will be digested
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? phage vector in cloning
Long (left) arm
short (right) arm
cos
Exogenous DNA (20-23 kb)
cos
short (right) arm
Long (left) arm
cos
cos
Exogenous DNA (20-23 kb)
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? replacement vector cloning
0.preparation of arm and genomic inserts
2. Packing with a mixture of the phage coat
proteins and phage DNA-processing enzymes

• Ligation

3. Infection and formation of plaques
Library constructed
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Gene libraries and screening
I 2 cDNA libraries
I2-1 mRNA isolation, purification
I2-2 Check theRNA integrity
I2-3 Fractionate and enrich mRNA
I2-4 Synthesis of cDNA
I2-5 Treatment of cDNA ends
I2-6 Ligation to vector
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I 2 cDNA libraries
cDNA libraries

• No cDNA library was made from prokaryotic mRNA.
• Prokaryotic mRNA is very unstable
• Genomic libraries of prokaryotes are easier to
  make and contain all the genome sequences.

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I 2 cDNA libraries
cDNA libraries

• cDNA libraries are very useful for eukaryotic
  gene analysis
• Condensed protein encoded gene libraries, have
  much less junk sequences.
• cDNAs have no introns ? genes can be expressed in
  E. coli directly
• Are very useful to identify new genes
• Tissue or cell type specific (differential
  expression of genes)

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I 2 cDNA libraries
I2-1 mRNA isolation
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I 2 cDNA libraries
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I2 cDNA libraries
Three methods to isolate mRNA.
1.Traditionally method was done by pass a
preparation of total RNA down a column of oligo
(dT)-cellulose

2.More rapid procedure is to add oligo(dT) linked
to magnetic beads directly to a cell lysate and
pulling out the mRNA using a strong magnet
3.Alternative route of isolating mRNA is lysing
cells and then preparing mRNA-ribosome complexes
on sucrose gradients
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I2 cDNA libraries
I2-2 Check the mRNA integrity
Make sure that the mRNA is not degraded.
Methods Translating the mRNA use cell-free
translation system as wheat germ extract or
rabbit reticulocyte lysate to see if the mRNAs
can be translated Analysis the mRNAs by gel
elctrophoresis use agarose or polyacrylamide
gels
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I2 cDNA libraries
I2-3 Cloning the particular mRNAs
Is useful especially one is trying to clone a
particular gene rather to make a complete cDNA
library.
Fractionate on the gel performed on the basis of
size, mRNAs of the interested sizes are recovered
from agarose gels
Enrichment carried out by hybridization Example
clone the hormone induced mRNAs (substrated cDNA
library)
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I2 cDNA libraries
I2-4 Synthesis of cDNA

First stand synthesis materials as reverse
transcriptase ,primer( oligo(dT) or
hexanucleotides) and dNTPs (Fig 1.1)
Second strand synthesis best way of making
full-length cDNA is to tail the 3-end of the
first strand and then use a complementary primer
to make the second. (Fig2.1)

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mRNA
I2 cDNA libraries
5
AAAAA-3
HO-TTTTTP-5
Reverse transcriptase Four dNTPs
mRNA
5
AAAAA-3
TTTTTP-5
3
cDNA
Terminal transferase dCTP
mRNA
AAAAA-3
5
TTTTTP-5
3-CCCCCCC
cDNA
Alkali (hydrolyaes RNA) Purify DNA oligo(dG)
5-pGGGG-OH
3-CCCCCCC
TTTTTP-5
cDNA
Klenow polymerase or reverse Transcriotase Four
dNTPs
5-pGGGG
-3
3-CCCCCCC
TTTTTP-5
Duplex cDNA
Fig 1.1 The first strand synthesis
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Duplex cDNA
5-pGGGG
-3
3-CCCCCCC
TTTTTp-5
Single strand-specific nuclease
5-pGGGG
-3
3-CCC
TTTTTp-5
Klenow polymerase
treat with E.coRI methylase
5-pGGGG
-3
3-CCCC
TTTTTp-5
Add E.colRI linkers using T4 DNA ligase
HO-CCG/AATTCGG-3
3-GGCTTAA/GCC-OH
HO-CCGAATTCGGGGGG
CCGAATTCGG-3
3-GGCTTAAGCCCCCC
TTTTTGGCTTAAGCC-OH
E.colRI digestion
5-pAATTCGGGGGG
CCG-3
3-CCCCCCC
TTTTTGGCTTAAp-5
Ligate to vector and transfom
Fig2.1 Second strand synthesis
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I2 cDNA libraries
I2-5 Treatment of cDNA ends
Blunt and ligation of large fragment is not
efficient, so we have to use special acid linkers
to create sticky ends for cloning.
The process

Move protruding 3-ends(strand-special nuclease)

Fill in missing 3 nucleotide (klenow fragment of
DNA polyI and 4 dNTPs)
Ligate the blunt-end and linkers(T4 DNA ligase)
Tailing with terminal transferase or using
adaptor molecules
Restriction enzyme digestion (E.coRI )
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I2 cDNA libraries
I2-6 Ligation to vector
Any vectors with an E.coRI site would
suitable for cloning the cDNA.
The process
Dephosphorylate the vector with
alkaline phosphatase

Ligate vector and cDNA with T4 DNA ligase
(plasmid or ? phage vector)
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Gene libraries and screening
I3 Screening procedures
I3-1 Screening
I3-2 Colony and plaque hybridization
I3-3 Expression screening
I3-4 Hybrid arrest and release
I3-5 Chromosome walking (repeat screening)
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I3 Screening procedures
I3-1 Screening
The process of identifying one particular clone
containing the gene of interest from among the
very large number of others in the gene library .

• Using nucleic acid probe to screen the library
  based on hybridization with nucleic acids.
• Analyze the protein product.

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I3 Screening procedures
Screening libraries
Searching the genes of interest in a DNA library

• Hybridization to identify the interested DNA or
  its RNA product
• Radiolabeled probes which is complementary to a
  region of the interested gene
• Probes
• An oligonucleotide derived from the sequence of a
  protein product of the gene
• A DNA fragment/oligo from a related gene of
  another species
• Blotting the DNA or RNA on a membrane
• Hybridize the labeled probe with DNA membrane
  (Southern) or RNA (Northern) membrane

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I3 Screening procedures
I3-2 Colony and plaque hybridization
Transfer the DNA in the plaque or colony to
a Nylon or nitrocellulose membrane
Phage DNA bind to the membrane directly
Bacterial colonies must be lysed to release DNA
on the membrane surface.
Hybridization (in a solution Containing Nucleic
acid probe)
(Alkali treatment)
X-ray film(radio- actively labeled )
antibody or enzyme (modified nucleotide labeled
Wash to remove unhybri- dization probe and
visualize
Line up the hybridizated region or repeated
hybridization
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I3 Screening procedures
Transfer to nitrocellulose or nylon membrane
Select positive from master plate
Keep master plate
Denature DNA(NaOH) Bake onto membrane
Probe with 32p-labled DNA complementary to
gene of interest
Expose to film
Screening by plaque hybridization
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I3 Screening procedures
I3-3 Expression screening

• Identify the protein product of an interested
  gene
• Protein activity
• Western blotting using a specific antibody

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I3 Screening procedures
Expression screening (1)
If the inserts are cloned into an expression
sites, it may be expressed. Therefore, we can
screen for the expressed proteins. However, this
screening may miss the right clone Example the
EcoRI site of lgt11 vector. The inserted genes
have one in six change (1/6) to be in both the
correct orientation (2 possibilities ? ) and
reading frame (three possibilities three
nucleotide code XXX).
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I3 Screening procedures
Expression screening (2)
Antibodies can be used to screen the expression
library.
The procedure has similarities to the plaque
hybridization
protocol.
Plaque lift ( taken by placing a membrane on
the dish of plaque)
Immersed in a solution of the antibody
Detected by other antibodies
Repeat cycles of screening to isolate pure
plaques
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????
1. Prokaryotic expression vector ?????? 2.
Baculovirus expression vector ?????????? 3.
Mammalian expression vector ???????? 4.
Adenoviral and retroviral vector ?????????????
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Prokaryotic expression vector ??????
GST-fusion 6xHis-fusion
GST
HIS
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??????

• Overexpression in cells ???,????
• RNAi ????
• Yeast two hybrid system ????????????????????
• Protein expression and antibody
  preparation?????????
• Localization of protein ?????????

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The end