pUC18: Plasmid, Cloning vector, Polylinker, [email protected]
Summary
TLDRIn this informative lecture, Dr. K Prime explores the widely recognized cloning vector pUC18, developed by the University of California. The talk delves into the nomenclature, features, and advantages of pUC18, highlighting its small size for accommodating larger DNA fragments, a high copy number for efficient amplification, and a multiple cloning site for versatile DNA insertion. The lecture also touches on the use of the pLac promoter for inducible expression and alpha complementation for visual detection of recombinants, providing a foundational understanding of plasmid-based cloning techniques.
Takeaways
- π¬ The lecture is about the pUC18 cloning vector, a well-known plasmid used in genetic engineering.
- π« The 'p' in pUC18 stands for plasmid, 'UC' stands for University of California, and '18' denotes its variant in the pUC series.
- 𧬠pUC18 is a cloning vector, not an expression vector, used for amplifying cloned genes in vivo.
- π Ideal cloning vectors like pUC18 are small in size, allowing for the cloning of larger DNA fragments.
- π pUC18 includes a pLac promoter from the lac operon of E. coli, which can be induced by IPTG to initiate transcription.
- 𧩠The vector contains a lacZ' gene encoding the alpha fragment of beta-galactosidase, which, when combined with the omega fragment from the host, forms a functional enzyme.
- π pUC18 has a multiple cloning site (MCS) with various restriction sites, enabling versatile DNA fragment cloning.
- π The vector includes an ampicillin resistance gene, beta-lactamase, allowing for the selection of transformed cells.
- π The origin of replication (ori) in pUC18, derived from the pMB1 vector, ensures the plasmid can replicate independently within the host.
- π pUC18 allows for visual differentiation between recombinants (white colonies) and non-recombinants (blue colonies) via alpha complementation in the presence of X-gal.
Q & A
What is the main topic of Dr. K Prime's lecture series?
-The main topic of Dr. K Prime's lecture series is the discussion of cloning vectors, specifically focusing on the most well-known cloning vector, pUC18.
What does the 'p' in pUC stand for in the context of plasmid nomenclature?
-In the context of plasmid nomenclature, 'p' stands for plasmid, indicating that the name refers to a specific type of plasmid.
What does 'UC' in pUC signify and what institution is associated with it?
-The 'UC' in pUC signifies the University of California, indicating that the plasmid was developed by researchers at this institution.
What are the two categories of vectors mentioned in the script?
-The two categories of vectors mentioned are cloning vectors and expression vectors.
Why are cloning vectors used in molecular biology?
-Cloning vectors are used to amplify the cloned gene of interest in vivo, providing a high copy number of plasmids that allow for the massive amplification of the inserted gene.
What is the size of the pUC18 vector and how does this size benefit its use as a cloning vector?
-The pUC18 vector is 2686 base pairs in size. Its small size is beneficial as it allows for the cloning of larger DNA fragments and is considered an ideal feature of a cloning vector.
What is the role of the pLac promoter in the pUC18 vector?
-The pLac promoter in the pUC18 vector is a site for binding with RNA polymerase, initiating transcription when induced by IPTG (isopropyl Ξ²-D-1-thiogalactopyranoside), which is an inducer of the Lac operon in E. coli.
What is the function of the lacZ' gene in the pUC18 vector and how does it relate to blue/white screening?
-The lacZ' gene encodes the alpha peptide of beta-galactosidase. It is used in blue/white screening, where the presence of an insert in the lacZ' region prevents the formation of functional beta-galactosidase, leading to white colonies, while non-recombinants form blue colonies due to the enzyme's activity with X-gal.
What is the MCS or polylinker in the pUC18 vector and why is it important?
-The MCS (Multiple Cloning Site) or polylinker in the pUC18 vector provides multiple restriction sites, allowing for the easy cloning of a variety of DNA fragments and enabling directional cloning without losing the open reading frame of lacZ'.
What is the purpose of the ampicillin resistance marker in the pUC18 vector?
-The ampicillin resistance marker serves as a genetic selection tool during transformation. It allows for the identification of E. coli that have successfully taken up the plasmid, as they will be resistant to ampicillin due to the presence of the beta-lactamase gene on the plasmid.
What is the role of the origin of replication (ori) in the pUC18 vector?
-The origin of replication (ori) is essential for the autonomous or independent replication of the plasmid. It is the site where replication is initiated, allowing the pUC18 to be a high copy number plasmid, which is beneficial for the amplification of the cloned DNA fragment.
What are the advantages of using pUC18 as a cloning vector over other vectors?
-The advantages of using pUC18 include its small size, which facilitates the cloning of larger DNA fragments and high transformation efficiency; its high copy number, which allows for the massive amplification of the cloned DNA; the presence of multiple cloning sites for directional cloning; and the ability for single-step visual detection of recombinants through alpha complementation.
Outlines
𧬠Introduction to pUC18 Cloning Vector
In this introductory lecture, Dr. K Prime discusses the pUC18, a well-known cloning vector. He explains the nomenclature of plasmids, highlighting that 'p' stands for plasmid and 'UC' refers to the University of California, where the vector was developed. The pUC18 is part of the pUC series and is a small, 2686 base pair vector, ideal for cloning larger DNA fragments due to its size. The lecture also covers the two types of vectors: cloning and expression vectors, with cloning vectors facilitating the amplification of the gene of interest within a high copy number plasmid. Dr. K Prime sets the stage for a deeper dive into the features and advantages of the pUC18 vector in subsequent lectures.
π¬ Features of pUC18: Plasmid Structure and Elements
This paragraph delves into the structural components of the pUC18 plasmid, which is a covalently closed, circular, double-stranded DNA molecule. The pUC18 is noted for its small size, which is crucial for cloning larger DNA fragments. The plasmid contains a pLac promoter, derived from the lac operon of E. coli, which can be induced by IPTG (isopropyl Ξ²-D-1-thiogalactopyranoside), allowing for the transcription of genes. The presence of the lacZ' gene, a fragment of the Ξ²-galactosidase gene, is also highlighted, which, when combined with the host's omega peptide, forms a functional enzyme. The paragraph further discusses the Multiple Cloning Site (MCS) or polylinker, which provides various restriction sites for easy and directional cloning of DNA fragments.
π‘οΈ pUC18 Cloning Vector: Resistance Marker and Replication Origin
The paragraph explains the importance of the ampicillin resistance marker in the pUC18 vector, which facilitates the selection of successfully transformed E. coli cells. The resistance is due to the presence of the Ξ²-lactamase gene, allowing cells to grow in the presence of ampicillin by breaking down the antibiotic. Additionally, the origin of replication (ori) is discussed as an essential element for the autonomous replication of the plasmid. The pUC18's ori is derived from pMB1, contributing to its high copy number, which is beneficial for amplifying the cloned DNA fragment for various downstream applications.
π Advantages and Applications of pUC18 Vector
The final paragraph outlines the advantages of using the pUC18 vector for cloning. Its small size enhances transformation efficiency and allows for the accommodation of larger DNA fragments, up to 7.5 kb. The presence of multiple restriction sites in the MCS enables the directional cloning of a variety of DNA fragments without losing any due to the small size of the insert. The paragraph also mentions the single-step visual detection of recombinants through alpha complementation, where non-recombinants form blue colonies due to functional beta-galactosidase activity, and recombinants remain white. Lastly, the potential for functional expression of the cloned gene using the inducible promoter is highlighted, allowing for the assessment of protein activity without massive protein production. The lecture concludes with an invitation for viewers to subscribe and engage with the channel for more informative content.
Mindmap
Keywords
π‘Cloning Vector
π‘Plasmid
π‘Nomenclature
π‘Promoter
π‘Lac Operon
π‘IPTG
π‘Multiple Cloning Site (MCS) or Polylinker
π‘Ampicillin Resistance Marker
π‘Beta-Lactamase
π‘Origin of Replication (Ori)
π‘Alpha Complementation
Highlights
Introduction to the most well-known cloning vector, pUC18.
Explanation of the nomenclature of pUC18, including the significance of 'p', 'UC', and the number 18.
Differentiation between cloning vectors and expression vectors, and their respective uses.
Description of the high copy number of pUC18 and its role in gene amplification.
The small size of pUC18, which allows for cloning of larger DNA fragments.
Introduction of the pLac promoter and its function in transcription initiation.
Use of IPTG as an inducer to activate the pLac promoter.
Role of the lacZ' gene in alpha complementation and blue/white screening.
Explanation of the multiple cloning site (MCS) or polylinker and its importance for cloning various DNA fragments.
Directional cloning made possible by the MCS in pUC18.
The ampicillin resistance marker for genetic selection after transformation.
Function of beta-lactamase in ampicillin resistance and its role in transformation screening.
Importance of the origin of replication (ori) for autonomous replication of the plasmid.
Advantages of pUC18 over other vectors, including its small size and high transformation efficiency.
Potential for cloning DNA fragments up to 7.5 kb in size with pUC18.
Visual detection of recombinants and non-recombinants through alpha complementation.
Functional expression of the cloned gene with the inducible promoter in pUC18.
Invitation to subscribe to Dr. K Frame's primer lecture series for more insights.
Transcripts
hello everyone
welcome back to dr k prem
primer lecture series
presenting by dr k prime that's me
today we will talk about
most well-known cloning vector that's
that is
18
18
so you all level of the
18 right
so that's a cloning vector
so you are
what is
let us look into the nomenclature of
pakistani
p stands for classmate that's always in
the any plasmid name if you see
the p is in the lower case
lowercase
then here
the
uc stands for university of
california this factor is developed by
developed by
university of california
and 18
is a num you know
variants in the park series
so there is a puck eight part
so that's a variant vectors
in the puck series
and here i'm saying it's a cloning
vector
vectors are two categories one is a
cloning vector and second one is a
expression vector
cloning vectors are used to
amplify the massive amplification of
cloned gene in in vivo
whenever you clone your gene of interest
into the cloning vector
that's a high copy
number of plasmid it will amplify
massively and you're going to get more
plasmid along with the plasmid your
inside also
amplified and you get it that
that your insert is amplified so
whatever purpose you know downstream
purposes may be pro preparation or
sub cloning you want to do the sub
cloning from cloning vector to
expression vector where you need more
amount of your
gene
or
or if in case of sequencing so that's a
that's why
always people starts with the cloning in
the learning vector then they'll move to
the expression vector
in this lecture i'm going to talk about
features of
packaging are elements of packaging and
their functions
and
what are the advantages of packaging
being a cloning vector
over the tbr return to two that is we
discussed in the previous uh lecture
right all right
let's move to the
elements of 18
so as you all know that plasmids are
covalently
closed
circular
extra chromosomal double strand dna
molecules right you see this is a
covalent requisite there is no open end
nicks are not there so covalently closed
circle it's a closed one
and circular extra chromosomal
right dna molecule double strand dna
molecule that they're called as a
plasmid this is an artificial plasmid
but 18 but 18 the size of this vector is
2686 base space
so smallest vector for a cloning purpose
so ideal
cloning vector should have two features
one of them is small size
small size
allows us to clone the bigger fragments
so ied first the feature is small size
of the vector is the
uh
ideal feature of a cloning vector and
this is a puck 18 fulfill that
first criteria
here the packet 18 have a
p lat that's a promoter
you all know promoters right promoters
are the
sites for
binding with the rna polymerase and then
once they bind to the promoter they
initiates the transcription
so this p lac promoter tilak promoter
is isolated from
lac operon of e coli you all know lac
operon right so lac operon is having uh
three genes structural genes one is a
beta electricity and transferase and
transporter right so this there on the
operon so that and the
lac operon
is
uh
on whenever there is a lactose and
whenever there is a absence of glucose
right so here the lag promoter is
isolated from the that operand and
inserted into the
uh bucket 18 vector
and
here
lag
is going to be induced by the
allo lactose
hello lactose that is nothing but
ippg ippz that's an inducer it will bind
to the pilat and allows it to bind to
the repressor of the lac operon
and allows the binding of rna polymerase
to the p lab and drives the transcript
drives the transcription
so ipt ipdg is a
ipd is a
isopropyl
beta galactoside that's a
non-metabolic
non-metabolic allo lactose means it'll
it is not metabolized it cannot be
metabolized by the system whatever
amount you add to the system it will be
there forever the same concentration
hence iptc is used to
switch on the lag promoter
black promoter right
under the downstream of the lag promoter
there is a small gene
that's called as lag z prime
lag z prime
so this is lag z prime is a
shortest
or you know shortened uh
short fragment of
beta galactosidase g
beta galaxy gene so as you all know that
beta galaxies beta galactosidase enzyme
which break downs the lactose into
glucose and the galactose right this is
having two
domains and terminal domain is called as
alpha peptide and c terminal domain is
called as omega peptide omega peptide so
the alpha fragment is from one to
one 140 amino acid and the rest is
almost like a 900 amino acid is the
omega if you chemically break down the
this enzyme into alpha and the beta
alpha and omega fragment and then you
again know club them they become a
functional protein functional protein so
here in this eoc 18 or pocket 18
large z that's a 13 nucleotides
encoding gene that's a lag z prime
so lag that prime is inserted downstream
to the promoter
promoter so whenever is iptc binds to
the uh repressor rna polymerase binds
binds to the electromotor and drives the
expression of laxative prime which
encodes the alpha peptide alpha peptide
and this alpha peptide and
an omega peptide in the host gene that's
in the oxygen omega from the ocean and
when you transform into the host e coli
is having the
omega fragment
and alpha fragment from the
vector
and omega fragment from the
host and they'll club and associate and
form a functional beta galacticity
right and use the
blue color colonies in the presence of x
dot that's i'll come to the next one so
this lag z prime
is
encodes the
alpha fragment of beta galactic space
in that
so that's a 30 uh 30 amino acid of
and beta
is and in the lag z prime region there
is a short region called as mcs mcs
multiple cloning site multiple cloning
site or polylinker so the these
sites provides the
more number of
more number of restriction sites
i can tell you you see
there is a eco r1
sst1 skp1
smart one damage one
one sol one
and uh you see psg one sph1 and indy3 is
so many almost one two three four five
six seven eight nine ten
we have a ten
restriction endonucleases in the
uh laxative primation that's inserted
without the losing the without losing
the open reading frame off for lag prime
so which allows the
cloning of variety of dna fragments
sometimes what happens uh your dna
fragment is having its mos site
you cannot use this mark
and if you have a equilibrium you cannot
use but you can use this bar a solvent
or n3 or bam h1 so it is 10 10 different
restriction sites are there in the mc
mcs or polylinker which will allows us
to clone the our dna fragment
very easily
and also
because of mccs we can clone our jin of
interest in a direction oriented for
example your
in your insert
if you're having a equal r1 at the
promoter region at the starting code
right and the endocodon is you are
taking the end three right so exactly
is start codon comes down under the p
lag promoter and
the end codon will goes to the
end of this flat set right so
directional flowing is also possible
with the
uh multiple cloning sites
right so you need to use the two
restriction enzymes to clone the your
gene of interest that is called as
directional cloning
the next element is
ampicillin resistance marker
amplisim resistance marker what is else
that's a genetic selection marker so to
store the transformations when you
transform the your vector and the answer
or that into the force
the
host which is which received the
uh plasmid
is resistant to
one of the antibiotics because the
your plasmid is carrying a
antibiotic resistance g here
ap stands for ampicillin resistance gene
what is that emphysema resistance gene
it's nothing but beta lactamase beta
lactamase beta-lactamase
is useful in the selection of for
transformations so whenever a
vector plasmid enter into the e coli
that e coli become resistant to the
amphitheater length then they can grow
in the presence of
civilians so the beta lactamase converts
the ampicillins or belongs to the
penicillin family
breakdowns the beta-lactam ring of
penicillins and converts into the
pencilic acid which is biologically
inactive
inactive so
the
genetic selection marker
ampersal resistant gene that is a
beta-lactamase is there in the packet 18
to screen the transformation
right
the last one not the least one that's a
origin of replication ori stands for
origin of replication
so origin of replication is very very
essential element of the
plasmid as well as the cloning vehicle
because
that is
enable the vector to be replicated
autonomously or independently
so autonomous replication or
independent replication of the chlorine
vehicle is all depends on the presence
of origin of replication from there
the replication is initiated
and this origin of replication is
uh collected from the pmb1 vector
and also npm this modification of the
origin of application is converted into
the high copy number plus high copy
number plasmid the puck 18 is a high
copy number plastic as i told you that
two important features of the cloning
vector that is a
small size which enables the
cloning of bigger dna fragments and the
second one is high copy number i copy
number so that the cloned dna fragment
is amplified more times massive
amplification so that you can harvest
and you can purify the plasmid and you
cut down and you can use the various
purposes
so
here
the bucket 18 is a
high copy number
classmate okay then in the next slide
i'm going to speak to you again the same
features in the text format right
advantages of over abr is equal
to
and you see
this is because of small size because of
small size it can ins it can
it can enter into the plasmid very
easily so that transformation rate is
also high so you get the more
transformation small size and the
transformation efficiency is more
small size vector can accommodate the
dna fragments of 10k as i told you that
plasmids are stable up to 10k
since it is 2686
instead of around 2.5
so you can clone another seven
and seven point five k or larger dna
you want to grow larger dna fragments
which are which are in size of seven to
seven point five maximum you can use
this vector
right right
then
multiple chlorine site or planet linker
provides the
many restriction enzyme sites
so that vector can be used to clone
variety of dna fragments and you can use
the two sides for the directional
cloning
we are not using you will not lose any
fragment because they are in the space
of
almost 20 to 30 nucleotides range only
right
and directional cloning is possible and
controls the orientation of your dna
fragment
and single step
visual
detection of recombinants
that's a recombinants will be in the
white color
white color colonies and
non-recombinants will be in the blue
color through the alpha complementation
when you clone your genov interest into
the
alpha
lux z prime region
and the the alpha peptide which is uh
coming from the
that
vector will not
associate with the
omega region from the omega peptide from
the host then what will happen
the recombinants recombinants
will not come if i will not have a
functional beta collector space and
which cannot metabolize the x-gal and
recap recombinants will be in a blue
color
and non-recombinants means where there
is no insert in the laxative prime
region which encodes the
usually uh alpha peptide that alpha
peptide can bind with the omega peptide
of host and forms the functional beta
galactosidase
and
the
non-recombinants or just transmits can
hydrolyze the
uh x gall
that's a
substrate to convert the
into a blue color
so
visual detection of non recombinants and
recombinants is possible with the part
18 vector so in the next uh
lecture i guess i will speak
exclusively on
what is alpha complementation i want
just uh i don't want to speak right now
in the next video speak but you know try
to see that also
and next one is functional expression of
the clone gene is possible with the
inducible promoter
so functional expression means it's not
it will not import the
high amount of protein but uh
it is possible to see
uh the functional experiments you can
essay the
that particular activity of the uh
protein if it is enzyme you can see the
uh you can see the engine activity from
the recombinant zone if it is a
transporter it accumulates the uh that
the substrate functional not uh like a
massive production of a protein is not
possible from this factor
so this is all about uh uh
you know 18
uh
elements and advantages of so if
at all you like it try to subscribe my
channel that is uh dr k frame
primer
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and
and if you have any comment please let
me know through the comment section i'll
get back to you quickly once i see them
thank you all for listening see you
again with one more video until then bye
bye
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