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Unit1. Basic concepts: DNA cloning.

In vitro
DNA recombination.
Recombinant DNA. Introduction of genetic information in bacteria
(transformation, conjugation and transduction)

Basic scheme of a cloning


process.
Basic steps in gene cloning experiment:
1. DNA fragment, containing the
gene to be cloned is inserted into a
circular DNA molecule called VECTOR to
produce a recombinant DNA molecule.
2. The vector acts as a vehicle that
transports the gene into a host cell,
which is usually a bacterium, although
others types of living cells can be used.
3. Within the host cell the vector
multiplies, producing numerous identical
copies not only of itself but also of the gene
that I carries.
4. When the host cell divides, copies of
the recombinant DNA molecule are passed to
the progeny and frther vector replication
takes place.
5. After a large number of cell divisions a
colony or clone of identical host cells is
produced.
Each cell in the clone contains one or more
copies of the recombinant DNA molecuke; the
gene carried by the recombinant molecule is
now said to be cloned.

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High-relevance discoveries: Bacterial plasmids and restriction


enzymes.
Plasmid

Restriction endonucleases enable DNA molecules to be cut at defined positions.


A restriction endonuclease is an enzyme that binds to a DNA molecule at a specific sequence
and makes a double stranded cut at or near that sequence. Because of the sequence
specificity the position of cuts within a DNA molecule can be predicted, assuming that the DNA
sequence is known, enabling defined segments to be excised from a larger molecule. This
ability underlies gene cloning and all other aspects of recombination DNA technology in wich
DNA fragments of known sequences are required.
There are three types of restriction endonuclease.
With types I and III there is no stric control over the
position of the cut relative to the specific sequence
in the DNA molecule that is recognized by the
enzyme. These enzymes are therefore less useful
because the sequences of the resulting fragment are
not precisely known. Type II enzymes do not suffer
from this, because the cut is always at the same
place, either within the example, the type II enzyme
called EcoRI cuts DNA only at the hexanucleotide 5
GAATTC 3. Digestion of DNA with a type I enzyme
gives a reproductible set of fragments whise sequences are predictable if the sequence of the
target DNA molecule is known.

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Unit 2. Plasmids and phages as cloning vectors. Artificial


chromosomes.
Recombination of DNA in vitro. Cut and paste with DNA molecules.

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Enzymes for the manipulation of nucleic acids: restriction


enzymes and other nucleases, ligases, enzymes that act on the 5
phosphate and polymerases.
The basis of recombinant DNA technology is the ability to manipulate DNA molecules in the
test tube. This depends on the availability if the purified enxymes whose activities are known
and can be controlled and which can therefore be used to make specified changes to the DNA
molecules that are being manipulated.
-

DNA polymerases. Enzumes that synthesize new polynucleotides complementary to an


existing DNA or RNA template.
Nucleases. Which degrade DNA molecules by breaking the phosphodiester bonds that
link one nucleotide to the next.
Ligases. Which join DNA molecules together by synthesizing phosphodiesrer bonds
between nucleotides at the ends of two different molecules or at the two ends of a
single molecule.
En-modification enzymes. Make changes to the end of DNA molecules adding an
important dimension to the desing of ligation experiments and providing one means of
labeling DNA molecules with radioactives and other markers.

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Some other Nucleases:


ExoIII
It has a 3' to 5' exonuclease activity which must initiate on blunt ended DNA or DNA with a 5'
overhang.
Exo VII
Exonuclease VII, (Exo VII) derived from E. coli, has a high enzymatic specificity for singlestranded DNA (ssDNA) and exhibits both 53 and 35 exonuclease activities. It is especially
useful for rapid removal of single stranded oligonucleotide primers from a completed PCR
reaction when different primers are required for subsequent PCR reactions.
Mung bean nuclease

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A single-strand specific DNA and RNA endonuclease which will degrade single-stranded
extensions from the ends of DNA and RNA molecules, leaving blunt, ligatable ends.
S1 nuclease
S1 Nuclease degrades single-stranded DNA and RNA endonucleolytically to yield 5phosphoryl-terminated products. Double-stranded nucleic acids (DNA:DNA, DNA:RNA or
RNA:RNA) are resistant to degradation except with extremely high concentrations of enzyme.

DNA POLYMERASES
Enzyme that synthesizes DNA is called DNA polymerase and one that copies an existing DNA or
RNA molecule is called template-dependent DNA polymerase.
The mode of action of a template dependent DNApolymerase
A template-dependent DNA polymerase makes a
new DNA polynucleotide whose sequence is
dictated, via the base-paired rules, by the sequence
of nucleotides in the DNA molecule that is being
copied.
New polynucleotide are synthesized in 53
direction.
DNA polymerase is unable to use an entirely single-stranded molecule as the template so in
order to start the synthesis there must be a short double stranded region to provide a 3 end
onto which the enzyme will add nucleotides.
In the test tube, a DNA copying reaction is
iniciated by attaching to the template a short
synthetic oligonucleotide (usually a about 20
nucleotides in length) that act as a primer for
DNA synthesis.
The position within the template molecule at
with DNA copying is initiated can be specified
by synthesizing a primer with the apropiate
nucleotide sequence.
(Apreciatte then the importance of priming in
PCR)

Many of these enzymes are multifunctional,


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being able to degradate DNA molecules as well


as synthesize them. DNA polymerases can also
have one or both of the following exonuclease
activities:
-

35 EXONUCLEASE. Nucleotides
removal from the 3 end of the strand
that has just been synthesized. This is
called PROOF-READING activity
because allow to correct errors by
removind incorrectly inserted
nucleotides.
53 EXONUCLEASE. Possessed by
DNA polymerases whose function in
genome replication requires that must
be able to remove at least part of
polynucleotide that is already attached
to the template strand that the
polymerase is copying.

DNA LIGASES.
DNA fragments that has been generated by treatment with a restriction endonuclease can be
joined back together again or attached to a new partner by a
DNA ligase.
The reaction requires energy (ATP or NAD).
The most used DNA ligase is obtained from
E.Coli cells infected with T4 bacteriophage.
It is involved in replication of the phage
DNA an is encoded by the T4 genome. Its
natural role us to synthesize
phosphodiester polynucleotide of a
double-stranded molecule.

In order to join together two restriction fragments, the ligase has to


synthesize two phosphodiester bonds one in each
strand.

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The reaction can ocure only if the ends to be joined come close enough to one another by
chance. If the who molecules have complementary sticky ends and the ends come together by
random diffusion, then transient base pair might form between the two overhangs. These base
pairs are not particulary stable but they may persist for sufficient time for ligase enzyme to
attach to the junction and synthesizes phosphodiester bonds to fuese the ends together.

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If the molecule are blunt ended then


they cannot base-pair to one another
not even temporaily, and ligation is less
efficient, even when the DNA
concentration is high and pairs of ends
are in relatively close proximity.
The efficiency of sticky end ligation has
stimulated the development of methods
for converting blunt ends into sticky
ends. Iin one method, short doublestranded molecules called linkers or
adaptors are attached to the blunt ends.
Linkers and adaptors work in slightly
different ways but both contain a
recognition sequence for a restriction
endonuclease with the apropiate
enzyme.

Another way to create a sticky end is by HOMOPOLYMER TAILING, in which nucleotides are
added one after the other to the 3 terminus at a blunt end. The enzume involved is called
terminal deoxynucleotidyl transferase.

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END-MODIFICATION ENZYMES.
Terminal deoxynucleotidyl transferase is a template-independent DNA polymerase because it
is able to synthesize a new DNA polynucleotide without basepairing of the incoming
nucleotides to an existing strand of DNA or RNA.
Alkaline phosphatas. Removes phosphate goups from the 5 phosphates can be ligated to one
another and a phosphatased end can ligate to a non phosphatased end but a link cannot be
formed between a pair of ends if neither carries a 5 phosphate. T4 polynucleotide kinase

T4 polynucleotide kinase

ATP => P-P-P-A, dnde el primer P es el P, el segundo P, es P y el tercer P es P. Si queremos


marcar una molcula, podemos hacerlo mediante la reaccin anterior, pero debemos tener en
cuenta que siempre se incorpora el P, por lo tanto debemos marcar siempre este, ya que los
otros dos permanecen en ADP.

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Unit 3. GENE TRANSFER IN ANIMAL CELLS: VECTORS.


E.Coli as a host cell for different vectors
Advantages:
1.
2.
3.
4.

Genetics and biochemistry well known


Stability of plasmids
Accepts several types of vectors
High production of recombinant proteins

Disadvantages:
1. Produces an endotoxine
2. Does not have splicing machinery
3. Post-translational modifications of proteins is different from that in eukaryotic cells.
Shuttle vectors have been developped
Different strains for different purposes
Easy to transform
Replicates vectors faithfully
Selectable markers available
DISCRIMINATION BETWEEN TRANSFORMED AND UNTRANSFORMED-CELLS.
1. DOMINANT SELECTION MARKERS:
AmpR: ampicillin acts on bacterial membrane

enzymes.

b-lactamase. Destroys ampicillin

CmR:

CAM: inhibits protein synthesis

(subunit 50S)

Cloramphenicol Acetyl Transferase.


In the presence of acetyl-CoA forms

TetR:

acetylated antibiotic-derivates

tet: inhibits protein synthesis (subunit

30S)

Membrane associated protein. Prevents the

entry of tetracycline to the cell

KanR: Kan: Inhibits protein synthesis


kanamyicil-acetyl-transferase.
Modifies the antibiotic.
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Description of some host-cell mutations which have proved to be very useful


end A: mutation in endonuclease I. Improves yield and quality of recombinant DNA preps
lacI [q]: overproduces lac repressor
Dlon: Deletion of lon protease. Reduces degradation of overproduced proteins (BL21)
dam: DNA adenine methylase. Mutation blocks methylation of adenine in GATC (DpnI, MboI,
Sau 3A, XbaI)
dcm: DNA cytosine methylase. mutation blocks methylation of cytosine in CC(A/T)GG

PLASMIDS
Plasmids are covalently closed double-stranded DNA molecules. Are circular molecules of DNA
that lead an independent existence in the bacterial cell. Plasmids almost always carry one or
more genes and often these genes are responsible for a useful characteristic displayed by the
host bacterium.
They behave as accessory genetic units; they replicate and inherit independently from the
bacterial chromosome.
They need the host cell for replication and transcription.
Often they provide genes that encode for enzymes which give some advantage to the host cell
under stressful environmental conditions.
In the laboratory antibiotic resistance is often used as a selectable marker to ensure that the
bacteria in a culture contain a particular plasmid.

Natural and artificial plasmids


Examples: Factor R; Factor F; pBR322; pUC; pBluescript .....
Modular structure
All plasmids possess at least one DNA sequence that can act as an origin of replication so they
are able to multiply within the cell quite independently of the main bacterial chromosome.
Modern plasmids contain a variety of features to aid the cloning process, including, for
example, polylinker regions containing many restriction enzyme sites.
A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which
contains many (up to ~20) restriction sites - a standard feature of
[1]
engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once
within a given plasmid. MCSs are commonly used during procedures involving molecular

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cloning or subcloning. Extremely useful inbiotechnology, bioengineering, and molecular


genetics, MCSs let a microbiologist insert a piece of DNA or several pieces of DNA into the
region of the MCS.

Todos los plsmidos contienen un adaptador de policlonaje (polilynker) con un amplio abanico
de dianas de insercin que se pueden usar individualmente o combinadas. En este ltimo caso
se puede definir la orientacin del insert. En muchos casos el fragmento de policlonaje est
situado en medio de un segmento derivado del opern lac de E.coli.
Selection marker are genes that add a new trait to cells.
Origin of replication, selection marker, MCS o Polylinker.........

Pbr322 is an artifial plasmid that contains 4,4kb.


It carries two antibiotic resistance genes . One
conferes resistance to ampicillin (Amp r) and the
other confers resistance ti tetracycline (tet r).
How does this vector works? Purified, closed
circular Pbr322 molecules are cut with a
restriction enzyme that lies within either of the
antibiotic resistance genes and cleaves the
plasmid DNA only once to create a single, linear,
sticky ended DNA molecules. These linear
molecules are combine with prepared target
DNA from a source organism. This DNA has been
cut with the same restriction enxyme, which
generates the same sticky ends as those on the
plasmid DNA. The DNA mixture is then treated
with T4 DNA ligase in the presence of ATP.
Under these conditions a number if different
ligated combinations are produced, including
the original closed circular plasmid DNA.

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(See also
pag:34)
The pUC series is derived from an earlier cloning
vector, Pbr322, which was originally constructed
by ligating together restriction fragments from
three naturally occurring E.coli plasmids.
pUC18 contanins an ampicillin resistance gene, a
regulatable segment of the B-galactosidae gene
(lacZ) of the lactose operon of E.coli. Ligation of a
new DNA into any one of these sites results in
insertional inactivation of the gene and hence loss
of b-galactosidase activity. This is the key to
distinguish a recombinant plasmid from a
nonrecombinant plasmid that has no new DNA.
Screening for B-galactosidase presence or absence
is in fact quite easy. (See Lac Selection)
LAC SELECTION:
Rather than assay for lactose being splite to
glucose and galactose, the presence of functional B-galactosidase molecules in the cell is
checked by a histochemical test with a compound called X-gal (a-complementation of bgalactosidase (X-gal: 5 bromo-4 chloro-3 indolyl-b-D-galactoside IPTG: isopropyl thio b-Dgalactoside)), which the enzyme converts into blue producuct. If x-gal (plis IPTG) is added to
the agar, along with ampicillin, then nonrecombinant colonies, the cells of which syntehize bgalactosidase will be colored blue whereas recombinant with a disrupted lacZ gene which are
unable to make b-galactosidase will be white.

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Substrain 71-18:
There is a lac operon deletion. Notwithstanding there is a F factor with a modified lac operon
(delta M15) allowing alpha-complementation.

Copy number control. Incompatibility


Copy number referes to the number of molecules of individual plasmid that are normally
found in a single bacterial cell. The factors that control copy number are not well understood
but each plasmid has a characteristic value that may be as low as one or as many as 50 or
more. Generally speaking, a useful cloning vehicle needs to be present in the cell in multiple
copies so that large quantities of the recombianat DNA molecules can be obtained.
REPLICATION AND INCOMPATIBILITY.
Several different kinds of plasmid may be found in a single cell , including more than one
different conjugative plasmid at any one time. In fact, cells of E.coli have known to contain up
to several diferent plasmids at once. To be able to coexist in the same cell different plasmids
must be compatible. If two plasmids are incompatible, one or the other will be lost from the
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cell.
If they share the ORI they are incompatible

There are several elements that works as replication origin in E.coli. You can have two
different plasmids in a single cell and grow them both if they contain selection markers. The
small transcript can join to the big one and the transcription turns off. So when we have hight
concentration of plasmid, there are acomulation of RNA and transcription stops.

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Bacteriophage lambda.
PROPERTIES, CYCLES LYTIC AND LYSOGENIC
Los bacterifagos son parasites bacterianos que solo pueden replicarse en el interior de una
bacteria. Sus funciones bsicas i de supervivencia son:
-

Proteccin de su cido nucleico del medio.


Introducci en el interior de una bacteria.
Conversin de la bacteria infectada en un sistema de produccin de partculas fgicas.
Liberacin al medio de los fagos completos.

Esta serie de sucesos constituyen el ciclo ltico. Los fagos que solo son capaces de llevar a cabo
este ciclo son conocidos como virulentos.
Los fagos atemperados (temperate) tienen como alternativa la integracin en el genoma del
husped y permanecen en lisogenia, es decir, replicndose con el cromosoma del husped y
expreando slo el gen represor, imprescindible para impedir que se inicie el ciclo titico.

El DNA de lambda es una molcula lineal de doble banda de unas 48,5KB. Se conoce su
secuencia totamente i en los extremos de la cadena posee un corto fragmento de 12 bases.
Estos expremos son llamados COS (Extremos cohesivos) son complementarios y permiten la
circularizacin del profago antes de la integracin en el genoma del husped.
Los genes localizados en la aparte izquierda del mapa lineal codifican las protenas de la cabeza
y cola, los de la aprte centrak estn implicados en la recombinacin y los procesos de
lisogenizacin, estos genes no son esenciales para la infeccin i crecimiento del fago por lo que
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pueden ser substituidos durante procesos de clonaje. Los genes de la derecha son los de
recgulacin e inmunidad (cI, cro, N)

La entrada de lambda en el husped apropiado produce la circularizacin de la molcla i la


transcripcin, con la RNApol del husped, a partir de los promotores PD i PI situados a derecha
e izquierda del represor cI .
Durante la fase inicial de la infeccin el fago circularizado se replica bidireccionalmente desde
un nico ORI que se activa por los productos de los genes O i P. Ms tarde empieza la
replicacin por el circulo rodante y se producen molculas lineales del profago cabeza-cola.
Estas molculas se cortan en los lugares cos por accin de la protena A (genes de la cabeza).
Cuando un bacterifago lambda tipo silvestre ubfecta bacteruas en un medio rico, solo una
prquea proporcin de las infecciones se abortar por el ciclo ltico para dar paso a la
integracin i lisogenia.
El llevar a cabo el ciclo ltico o lisognico depende de un comple equilibrio entre una serie de
productos gnicos del fago y del husped. Los niveles del producto cI tienen una importancia
critica en la eleccin de la via ltica o lisogenica. Si son altos el represor gana i se establece la
lisogenia, si son bajos, la via ltica.
Cloning: Phages give rise to plaques
Selection : plaques indicate infection. There are strategies to identify insert containing vectors/plaques.
Cuando un fago infecta una bacteria libera un gran numero de particulas fagicas que, a su vez, inyectan
las bacterias de alrededor. El resultado es una zon aclara em un csped (turbio) de bacyerias q se
denomina calva (plaque).

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La identificacin de una lisis plaque cuyos fagos lleven un gen de inters entre todas las plaques de lisis
crecidas se suelen realizar mediante mtodos de hibridacin.

Inserts: up to 25 Kb
Introduction of DNA in host cells: nude DNA and infection with in vitro reconstituted virus
High recuperation: up to 106-107 phage particles per plaque.
High sensitivity of detection with hybridization probes.

El fago lambda wild type no es adecuado para su utilizacin como vector ya q tiene
numerosas dianas de restriccin para cualquiern enzima y baja capacidad sin eliminar
parte del genoma propio para insertos de gran tamao. Por ello hay variantes aptas
para el clonaje, que han sido modificadas eliminando dianas o colocndolas en luhares
idneos (Izquierdo, p129):
-

Insertion vectors: Part or all the optional DNA has been removed an a unique
restriction site introduced at somo position within the trimmed down genome.
Made by the direct integration of the insert in a host target sequence.

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Permite cloner pequeos fragmentos de cDNA enla regin de cI. Por tanto los recombinantes
sern cI- . Los bacterifagos sin el DNA heterologo harn la lisogenesi i no formaran plaques.

Replacement vectors: The optional DNA is contained, witgin a stuffer fragment,


flanked by a pair of restriction sites, that is replaced when the DNA to be cloned is
liggated into the vector. Son aquellos en los que el segmeno n esencial del fago es
reemplazado por un insert de DNA heterologo.
Substitution vectors gas a stuffer fragment that has tarfet sequences for restriction
enzymes so it makes easier the addition of our insert

Poseen dos regions de policlonage a ambos lados de una region de rlleno procedente de E.Coli
y que sera subtituida por un insert. Permite hasta unas 20kb, utilizando cualquiera de las
dianas de restriccin orientadas en sentido opuesto a ambos ados de las sequencias de
relleno.

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In vitro DNA PACKING

The lambda genome is linear but the two natural ends of the molecile have 12-nucleotidese
single stranded overhands (COS SITES)which have complementary sequences and so can base
pair to one another. A lambda cloning vector can therefore be obtained as a circular molecule
chich can be manipulated in the same way as a plasmid and re-introduced into E.coli by
transfection, the term used for uptake of naked phage DNA. Alternatively a more efficient
uptake system called in vitro packinh can be used. This procedure starts with the lineal version
of the cloning vector, the initial restriction cutting the molecule into two segments, the left
and the right arms, each with a cos site at one end. The ligation is carried out with quatities of
each arm and the DNA ti be cloned, the aim beig to produce concatamersn in which the
different fragments are linked together in the order left arm new DNA- right arm. The
concatamers are the added to an in vitro packing mix wich contains all the proteins needed to
make a lambda phage particle. The proteins form phage particles spontaneously and will place
inside tge particles any DNA fragment that is between 37-52kb out of concatamers and
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construct lambda phages around them. The phages are the mixed with E.coli cells and the
natural infection process transports the vector + new DNA into bacteria. (Genomes 2, pg 113)

GENOMIC DNA LIBRARY


Librera genmica: Conjunto de clones en el que est representado todo el genoma de un
organismo. El tamao depender del genoma y el tamao medio de los fragmentos
individuales (insertos).
Los pasos para construirla seran los que se muestran en la figura:

ADN genmico y de alto peso molecular se trata con encimas para obtener un tamao medio
de DNA. Se perepara a su vez ADN del fago lambda para ser utilizado como vector de
sustitucin. El ADN del vector se trata con tambin con enzimas. Hhay q ser cuidadoso de crear
extremos cohesivos para que puedan unirse ambos extremos por complementariedad de las
secuencias. Se substituye un fragmento del vector por uno de inserto, lo que nos permite

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recuperar el tamao del genoma del fago original. El fago lambda solo empaqueta ADN de
cierto tamao, con lo que quedarn excluidos aquellos que unan brazos fgicos sin inserto.
La enzima ligasa unir covalentemente los extremos apareados por las dianas de restriccin y
los fagos se empaquetaran in vitro. El fago lambda normalmente se replica por el mtodo del
crculo rodante y empaqueta su ADN cortado en las regiones Cos. La regin Cos, cuando es
cortada da lugar a terminales cohesivos de 12 pb que constituirn los extremos del profago.
Para el paquetamiento in vitro de libreras genmicas se utiliza frecuentemente un mutante
del fago lambda deleccionado en las secuencias cos. Este mutante se mantiene en dorma
lisognica (como profago) en una cepa de E.Coli. Al introducir el ciclo ltico se origina una
acumulacin intracelular de todos los componentes del empaqietamiento, inclyendo las
precabezas vacias. El paso siguiente est bloqueado. Lisados de este mutante se pueden aadr
a ADN recombinante que posea secuencias Cos. De este modo, solo el ADN recombinado ser
empaquetado en el fago.

Deteccin del clon deseado.


Cuando un fago infecta una bacteria libera un gran nmero de particulas fgicas que infectan
las bacterias de alrededor. El resultado es una zona clara y un csped (turbio) de bacterias que
se denomina calva (plaque).
La identificacin de la plaqu de lisis fagica cuyos fagos lleven un gen de inters de entre todas
las plaques de lisis crecidas sobre el csped de bacterias o la de una colonia bacteriana de
entre todas las crecidas en una placa petri es una prctica comn.
Pagina 108, Izquierdo.

Lambda lisogen Cell with provirus integrated

COSMIDS
Plsmidos a los que se les ha insertado la regin COS del fago lambda. Son partiularmente
tiles para clonar insertos de gran tamao. COS confiere al plasmido la facilidad para ser
empaquetado in vitro por l sistema del fago siempre que existan de 35-52kb entre COS y COS.
Lo que implica una seleccin efectiva para insertos de entre 40-45kb.
Una vez l fago haya inyectado el DNA en la bacteria este se comportara como n plsmido y
podr ser seleccionado por resistncia a ampicilina.

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PREPARATION OF LAMBDA DNA


What is a lysogen?
What do you think inducing a lysogen is?
Where does the phage used to derive the BHB2688 or
BHB2690 come from?
Sup+ strains

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Filamentous phages. Characteristics


Filamentous phages (M13, fd y f1)
Circular single-stranded DNA of 6407 bp
They infect E.Coli F+, F or HFR
Tubular shape. Coat formed by 2700 units of the gene 8
product. There are other much less abundant proteins
in the coat.
There seems to be no size packaging limit. The longer
the genome, the larger the particle.
Phage DNA can be isolated as ssDNA or as dsDNA
They were very useful for DNA sequencing and sitedirected
mutagenesis.
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The genome encodes 10 proteins and there is an intergenic region (IG) which contains the
origins of replication for both strands, + and -, the packaging signal for the + strand and a
transcription termination sequence.

The M13 ORI allows the

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production of ssDNA for fragments cloned in plasmids which contain the ORI.

Required phage factors are provided by helper phages Helper phages have defects in their
replication origin that result in a poor growth, but they express all the transrequired elements
for production of phages
Bacterifago M13
Es un fago filamentoso, su DNA es circular de hebra sencilla y 6,4kb. Infecta bacterias
portadoras de un episoma F a travs de los filamentos sexuales F (pili). La replicacin del fago
no produce la lisi de la bacteria sino que esta continua creciendo lentamente excretabdi
particular al medio.
El DNA viral tiene una fase replicativa en la que se originan unas 100 copiasde DNA
nicartenario. Posteriormente se acumulan unas protenas de unin al DNA de banda simple
codificadas por el fago, que impiden la sintesi de l cadena complementaria, la maduracin
tiene lugar en la membrana, donde se sustituyen las protenas de unin a ADN de hebra
sencilla por las de la capside.
En los procesos de clonacin es mas fcil trabajar con ADN bicatenario. sta es la razn por la
que se utiliza preferentemente la forma replicativa de M13, esta forma se puede aislar de
clulas infectadas, manipular como cualquier ADN de plsmido y reintroducir en clulas por
transformar. El ADN de doble banda se incorporar al ciclo de replicacin y generar aprtculas
fagicas que contendrn solo una de las dos hebras del ADN + i la otra nunca se empaquetar.
Tanto el ADN de la forma replicativa como el de hebra sencilla del fago pueden transfectar
clulas competentes de E.Coli y proseguir con el ciclo vital.
No hay grandes restricciones respecto al tamao del inserto puesto que el tamao final de la
particula viene determinado por la longitud del ADN, sin embargo los los insertos grandes son
muy inestables y acaban por ser eliminados o acortados.

Fagemids (pUC, pBluescript series)


Mitad fago mitad plsmido. Se obtienen comerciamente. El clonae en fagomidos es
particularmente atarctivo para secuenciar por el mtodo de didexidos i para realizar
mutagnesi dirigida.
Bluescript lleva el origen de replicacin de pUC y los primero 146 aminocidos del gen lac Z
que por comeplemntacion intra-alelica rescata una mutacin en el mismo gen del husped (al
inicio) y permite seleccionar por colonias coloreadas cuando se utiliza como plsmido.
En estos vectores todos los genes M13 son esenciales por lo que se necesita de un fago
auxiliar que facilite en trans las funciones eliminatorias. A cada extremo de la regin de
policlonaje se encuentra el promotor T7 o T3 en sentidos opuestos para poder transcribir el
inserto en cualquier orientacin que entre. En ausencia de un fago auxiliar M13 o f1 los
fagmidos son indistingibles de los plsmidos, pero en presencia de ellos se activa el ORI
fgico y se produce ADN monocatenario y se exportan viriones al medio.
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Estos viriones sern de dos tipos: recombinantes y auxiliares. En la mayora de los casos no
ser necesario separar los unos de los otros ya q se emplearan primers especficos que solo
estn presentes en los recombinantes.

Helper phage

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Purification of ssDNA and replicative forms

CLONING IN YEAST
Production of proteins with a commercial value:
Post translational.protein modification
No toxins
Expression with genomic DNA
Large mutant collection available: facilitates detection and isolation of recombinants
Small genome (1.4 X10E7 versus 3X10E9)
Rapid growth (<2 hours to divide). They can grow in suspension or form colonies on agar
Division by sexual or asexual reproduction
Can be grown as haploids or diploids facilitating the isolation of recessive mutations
Yeast have a polysaccharide wall around the cell membrane which hampers DNA uptake.
Las levaduras mantienen las ventajas de los organismos en cuanto a que son unicelulares, de
facil manipulacin y crecimiento rpido. Su organismo celular es eucariota lo que permite la
realizacin de procesos de expresin y maduracin caracteristicos de las clulas animales i
vegetales.
Son capaces llevar a cabo prcesos que no realizan las bacterias y que pueden ser importantes
para la expresin de un gen eucaritico en un sistema heterlogo. Realizan acetilaciones Nterminales, N-glicosilaciones, fosforilaciones, eliminacin de la metionina inicial de la protena
madura, secrecin proteica, procesamientos proteoltico, permiten unin y ensamblaje de
subunidades en macromolculas.

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Facil y baratas de cutivar.


Permiten clonar fragmentos de DNA de gra tamao. 100-2000kB de cualquier organismo
pueden ser aislados en vetores YAC. Esto es interesante cuando se quiere aislar un gen
completo que posee muchos intrones y est muy extendido en el genoma original.

Spheroplast transformation.
To transform cells, spheroplasts are produced by removing the wall enzymatically or by
treatment with lithium acetate
Spheroplasts behave as competent bacteria and can take DNA from the environment in the
presence of CaCl2. The cell wall can be synthesized again in the appropriate growth medium.
Large DNA fragments can be cloned in YACS

En levaduras, podemos distinguir dos tipos de portadores gnicos:


Vectores de integracin cromosmica : Tienen un plsmido procaritico y secuncias
de levadura. Estas ltimas permiten una integracin en su propio genoma por
recombinacin homloga. Se utilizan principalmente para introducir un gen de inters
en la levadura, i para provocar delecciones en genes concretos por recombinacin
homloga.
Vectores de replicacin autnoma (YAC, 2)

Markers: auxotrofism and antibiotic resistance.

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Auxotrophy (Gr. "to increase"; "nourishment") is the inability of an organism to


synthesize a particular organic compound required for its growth (as defined by IUPAC). An auxotroph is
an organism that displays this characteristic; auxotrophic is the corresponding adjective. Auxotrophy is
the opposite of prototrophy, which is characterized by the ability to synthesize all the compounds
needed for growth.
Replica plating is a technique in which one or more secondary Petri plates containing different solid
(agar-based) selective growth media (lacking nutrients or containing chemical growth inhibitors such
as antibiotics) are inoculated with the same colonies of microorganisms from a primary plate (or master
dish), reproducing the original spatial pattern of colonies. The technique involves pressing a velveteencovered disk, and then imprinting secondary plates with cells in colonies removed from the original
plate by the material. Generally, large numbers of colonies (roughly 30-300) are replica plated due to
the difficulty in streaking each out individually onto a separate plate.
The purpose of replica plating is to be able to compare the master plate and any secondary plates,
typically to screen for a desired phenotype. For example, when a colony which was present on the
primary plate(or master dish), fails to appear on a secondary plate, it shows that the colony was
sensitive to a substance on that particular secondary plate. Common screenable phenotypes
include auxotrophy and antibiotic resistance.
Replica plating is especially useful for "negative selection". However, it's more correct to refer to
"negative screening" instead of using the term 'selection'. For example, if one wanted to select colonies
+
that were sensitive to ampicillin, the primary plate could be replica plated on a secondary Amp agar
plate . The sensitive colonies on the secondary plate would die but the colonies could still be deduced
from the primary plate since the two have the same spatial patterns from ampicillin resistant colonies.
The sensitive colonies could then be picked off from the primary plate. While doing this, frequently the
last plate will be non-selective, in this example, a nonselective plate will be replica plated after the
Amp+ plate, to confirm that the absence of growth on the selective plate is due to the selection itself,
and not a problem with transferring cells. Basically, if one sees growth on the third (nonselective) plate
but not the second one, this indicates the selective agent is responsible for the lack of growth; if the
non-selective plate shows no growth then one cannot say whether viable cells were transferred at all

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and no conclusions can be made about the presence or absence of growth on selective media. This is
particularly useful if there are questions about the age or viability of the cells on the original plate.

Integrative and replicative plasmids (ARS elements).


An autonomously replicating sequence (ARS) contains the origin of replication in
the yeast genome.

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Stability of transformants (CEN sequences).

Los vectores ARS/CEN son vectores ARS a los que se les ha aadido secuencias centromricas
para augmentar su estabilidad. La regin centromrica es responsable de la secrecin de las
clulas hijas al crear una estructura cromatnica especial. No pa

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YACs

Los vectores YAC (yeast astificial chromosome) son vectores ARS/CEN a los que se les ha
aadido telmeros cromosmicos. Permiten clonar los fragmentos ms grandes de DNA
siendo especialmente tiles cuando interesa aislar genes con muchos intrones que ocupan una
gran extensin del genoma.
Incluyen un plsmido procaritico para su propagacin y preparacin en E.Coli. Se puede
cortar con EcoRI en dos fragmentos que constituyen los brazos cromosmicos. El brazo
izquierdo tiene un telmero, un centrmero, una secuncia ARS i un gen de resistncia a
ampicilina para la seleccin. El brazo derecho solo tiene otro telmero y otro gen de seleccin
URA3.
Se emplea principalmente para elaborar libreras de genomas muy grandes .

El cromosoma artificial de levaduras o YAC (Yeast artificial chromosome) forma parte de los vectores de
clonacin de alta capacidad siendo, de hecho, el de mayor capacidad (200 kb - 3.000 kb). Fueron
descritos por primera vez en 1983 por Murray y Szostack.

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Es un vector que pretende imitar las caractersticas de un cromosoma normal de


una levadura (eucariota), portando un centrmero y telmeros terminales. Esto permite clonar en
levaduras (microorganismos eucariotas) secuencias de ADN de hasta un milln de pares de bases o ms,
al comportarse como un cromosoma propio de la levadura.
Son utilizados en construccin de genotecas genmicas, siendo muy extendido su uso en los primeros
aos del Proyecto Genoma Humano. Sin embargo, son ms inestables que otros vectores como BACs
(cromosoma artificial bacteriano), que han acabado imponindose.
Los YAC tienen un origen de replicacin relajado en procariotas, por lo que aparecen muchas copias en
cada bacteria (gran utilidad para producir masivamente el vector), pero un origen de replicacin estricto
en levaduras, apareciendo slo una copia por clula de levadura.
Un YAC tpico tiene:

ARS1: secuencia de replicacin autnoma


CEN4: centrmero del cromosoma IV, permite la segregacin del plsmido durante la divisin
celular
TELs: secuencias telomricas.
HIS3, TRP1, URA3: marcadores de seleccin
en levadura confieren prototrofa para histidina, triptfano y uracilo
SUP4: cambia la mutacin sin sentido mbar (UAG) por la incorporacin de tirosina. Identifica las
clulas que incorporan el vector, ya que las clulas husped que portan esta mutacin mbar, de
color rosado, pasan a tener un fenotipo blanco. (Prototrofo para ade).

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Homologous recombination

Per determiner la function de un gen, a veces se puede producer una mutacin y ver el efecto
que ello genera en la clula o organismo. Permite producir mutacione sin vivo a nivel del gen.

GENE KNOCK OUT.


Para eliminar la function de un gen de la levadura por una Diana gnica utilizaremos la
recombinacin homloga, que sustituir el gen endgeno por la versin deleccionada e
interrumpida.

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Mutantes letales tambien pueden ser rescatados en levaduras iploides donde al inducir la
esporulacin en un mediopobre, habr igual proporcin de esporas haploides letales como
normales

.
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