Plasmid DNA vector in gene cloning | plasmid vector | pbr322 vector | puc 19 vector
Summary
TLDRThis educational video script delves into the world of plasmid vectors, a crucial component in recombinant DNA technology. It explains plasmids as self-replicating, circular DNA molecules used for gene delivery and amplification, particularly in E. coli bacteria. The script highlights two common plasmid vectors, pBR322 and pUC19, discussing their naming, components, and functions. It further clarifies the distinction between cloning and expression vectors, the importance of insert size, and the features essential for a DNA molecule to serve as a vector. The advantages of plasmid vectors, such as ease of handling and high copy numbers, are contrasted with their limitations, mainly the maximum insert size they can accommodate.
Takeaways
- 🧬 Plasmid vectors are essential tools in recombinant DNA technology for gene delivery and amplification.
- 🔄 Plasmids are self-replicating, circular, double-stranded DNA molecules that can be used for cloning and expression purposes.
- 🔍 Plasmid vectors must have an origin of replication (ori), a multiple cloning site (MCS), and a selectable marker for successful cloning.
- 📏 Plasmid vectors typically have an insert size limitation, ranging from 5 to 25 kilobases (kb), depending on the vector type.
- 🚀 Two common plasmid vectors mentioned are pBR322 and pUC19, each with specific features and uses.
- 📦 Plasmids are advantageous due to their small size, ease of handling, and large copy numbers within host cells.
- 🔬 Plasmids are ideal for cloning small DNA fragments and can also be used for gene expression if equipped with a promoter region.
- 🛑 The main disadvantage of plasmid vectors is their limited insert capacity, which restricts the size of the target gene that can be cloned.
- 🔬 The script discusses the components of plasmid vectors, including origin of replication, selectable markers (often antibiotic resistance genes), and multiple cloning sites.
- 🔑 Selectable markers are crucial for distinguishing between recombinant and non-recombinant plasmids, aiding in the selection process.
- 🔬 The script also mentions the importance of the copy number of plasmids within a host cell, which can vary and affect the efficiency of cloning.
Q & A
What is a plasmid vector in the context of recombinant DNA technology?
-A plasmid vector is a self-replicating, circular double-stranded DNA molecule used as a vehicle to deliver and amplify a target gene within a host cell, commonly used in molecular cloning and gene expression.
What are the two main types of vectors mentioned in the script?
-The two main types of vectors mentioned are cloning vectors, which deliver the target gene into the host cell for amplification, and expression vectors, which allow the target gene to be transcribed and translated into a protein.
Why are plasmid vectors named with specific codes like pBR322 or pUC19?
-The names pBR322 and pUC19 are specific identifiers for particular plasmid vectors, which may reflect certain characteristics or features of the plasmid, such as its size, origin, or resistance markers.
What is the significance of the copy number in plasmid vectors?
-The copy number refers to the number of plasmid copies that can be present within a single host cell. It is significant because it affects the efficiency of gene amplification, with higher copy numbers generally leading to more efficient amplification.
What are the essential components of a plasmid vector?
-Essential components of a plasmid vector include an origin of replication (ori), a selectable marker (often an antibiotic resistance gene), multiple cloning sites (also known as polylinker or MCS), and a promoter region if used as an expression vector.
What is the insert size limitation for plasmid vectors?
-The insert size for plasmid vectors generally ranges from 5 to 25 kilobases (kb), with specific vectors like pUC19 having a maximum insert size of 15 kb.
What is the purpose of the selectable marker in a plasmid vector?
-The selectable marker, often an antibiotic resistance gene, is used to distinguish between recombinant plasmids containing the target DNA and non-recombinant plasmids, allowing for the selection of successfully modified plasmids.
What are the advantages of using plasmid vectors for molecular cloning?
-Plasmid vectors are advantageous due to their small size, ease of handling and purification, efficient selection and screening processes, and their natural self-replication within bacterial hosts, making them ideal for cloning small DNA fragments.
What is the main disadvantage of using plasmid vectors for cloning larger DNA sequences?
-The main disadvantage is the limited insert size that plasmid vectors can accommodate. Attempting to clone larger sequences can lead to issues with recombinant plasmid formation, maintenance within the host cell, and reduced transformation efficiency.
Can you provide an example of a plasmid vector mentioned in the script?
-An example of a plasmid vector mentioned in the script is pUC19, which has specific features such as an origin of replication, a promoter, and antibiotic resistance sites, making it suitable for both cloning and expression purposes.
Outlines
🧬 Introduction to Plasmid Vectors in Recombinant DNA Technology
This paragraph introduces the concept of plasmid vectors, a crucial component in recombinant DNA technology. It explains that plasmids are circular, self-replicating DNA molecules used for gene delivery and amplification, particularly in molecular cloning. The paragraph highlights the importance of understanding plasmids for those with a background in biotechnology, microbiology, or molecular biology. It also distinguishes between cloning vectors, which deliver the target gene into the host cell for amplification, and expression vectors, which facilitate the transcription and translation of the target gene into a protein. The pVR322 and pUC19 vectors are mentioned as widely used examples, setting the stage for a deeper dive into their characteristics and components.
🔬 Components and Characteristics of Plasmid Vectors
This paragraph delves into the specific components and characteristics that define a plasmid vector. It emphasizes the necessity of an origin of replication for self-replication, a multiple cloning site for insertion of the target DNA, and a selectable marker to differentiate between recombinant and non-recombinant plasmids. The paragraph also discusses the insert size limitation, which typically ranges from 5 to 25 kilobases for plasmid vectors, with examples like pUC19 having a maximum insert size of 15 kilobases. The summary includes the advantages of using plasmid vectors, such as their small size, ease of handling, and high copy numbers within host cells, and touches on the disadvantages, primarily the limited insert size capacity, which can affect the cloning of larger DNA sequences.
📈 Advantages and Disadvantages of Plasmid Vectors
The final paragraph wraps up the discussion on plasmid vectors by summarizing their advantages and disadvantages. It points out that plasmids are ideal for cloning small DNA fragments due to their self-replicating nature and ease of maintenance within bacterial hosts, which can accommodate a high copy number of plasmids. However, it also notes the limitation of plasmids in handling larger DNA sequences, as attempting to clone sequences over 15 kb can lead to issues with recombinant plasmid formation and reduced transformation efficiency. The paragraph concludes by setting the stage for further exploration of specific plasmid vectors like pBR322 and pUC19, including their naming, modifications, and comparative advantages and disadvantages.
Mindmap
Keywords
💡Plasmid Vector
💡Recombinant DNA Technology
💡Cloning Vector
💡Expression Vector
💡Self-replicating
💡Insert Size
💡Selectable Marker
💡Multiple Cloning Site (MCS)
💡Origin of Replication (ori)
💡Copy Number
💡PBR322 and pUC19
Highlights
Introduction to plasmid vectors in recombinant DNA technology.
Explanation of plasmid vectors as gene delivery systems.
Differentiation between cloning and expression vectors.
The necessity of self-replication capability in vectors.
Description of plasmids as circular, self-replicating DNA.
Importance of copy number in plasmid vectors.
Properties required for a DNA to be considered a vector.
Insert size limitations of plasmid vectors.
Components of a plasmid vector including origin of replication and selectable markers.
Role of multiple cloning sites or polylinkers in vector manipulation.
Differentiation between recombinant and non-recombinant plasmids using selectable markers.
Advantages of plasmid vectors such as ease of handling and high copy numbers.
Disadvantages of plasmid vectors, primarily the limited insert size.
Practical applications of plasmid vectors in molecular cloning and gene expression.
Introduction to specific plasmid vectors: pBR322 and pUC19.
Discussion on the naming conventions of plasmid vectors.
Comparative analysis of the advantages and disadvantages of pBR322 and pUC19 vectors.
Transcripts
all right everyone this is a time to
talk about another important chapter and
that is plasmid vector so of course we
are talking about the recombinant dna
technology and we're talking about
different kinds of vectors and this time
we are going to talk about plasmid
vector an under plasmic vector we are
also going to talk about two important
kind of vector widely used plasmid
vectors like pvr 322 vector and puc19
vector we also see why they are named
like pbr 322 or puc19 and what are their
components but before that understand
about the plasmid vector plasmid is a
term that you must heard if you are from
biotechnology background or microbiology
background or any molecular biology
biochemistry background it's very common
term now why and what you mean by
plasmic vector basically plasmid vector
is what is a vector a vector is a gene
delivery system a gene delivers is a
part of a gene delivery system so
basically when we do molecular cloning
the process of amplifying the number of
a target gene okay when so we need we
need a target gene we need the target
gene to be amplified under a host let's
say the host is e coli bacteria fine so
e coli bacteria is the host the target
gene is identified now the target gene
needs to be inserted somewhere who will
go inside the host cell and can be can
be participating in the process of
amplification which is the replication
of
the whole
target gene including
the vehicle where we are inserting the
target gene to be amplified so the
vehicle which is carrying our target
gene for amplification is known as
vector okay that is the cloning vector
so there are two kinds of vector cloning
vector and expression vector cloning
vector is the vector with which we can
deliver the target gene inside the host
cell and it will grow inside the whole
cell in number while in expression
vector are those vector where the target
gene can be transcribed and translated
into the target protein that's why you
call it expression gene expression okay
so this is plasmid vector is in this
case a cloning vector or it can be a
cloning vector it can be expression
vector but in this case we are talking
about the cloning process so the vector
is a vehicle with which we're delivering
our target
dna target gene
so
this plasmid vector is one of such kind
of delivery vehicle for the target gene
to be inserted so for a vector what we
need is that a vector must have the
capability of self replicating
capability of self replication it's very
very important so plasmid what is
plasmid it's a circular dna
self replicating a circular self
replicating dna so this plasmid circular
self replicating double stranded dna
which is present inside the host cell
which can be transferred inside the host
cell
and the host is a bacterial cell let's
say equalize cell so once the vector is
inside the e coli cell it can grow in
number okay it can grow in number
and not only that but there is a number
of cells number of vector the number of
plasmid can be present inside the host
cell that is known as a copy number in
some case it can be 50 60 some case 200
sub case 300 some case 600 some case
1000 so there are different copy numbers
of a plasmid okay the smaller the
plasmid the higher chance that it can be
accommodated inside of the higher
numbers it can be accommodated inside of
the host cell
so for a vector there are some
properties needed to call
a circular dna or a linear dna to be a
vector okay so what a vector should
carry it should carry a insert
insert size insert means what the target
dna so it should have a capacity to
carry a target dna or dna of our
interest that is insert so it has an
insert size limitation depending upon
its own size also it has an insert size
limitation so the bigger the plasmid is
uh the bigger the vector is the higher
the insert size can become okay so
plasmid vectors are smaller so their
insert size is also smaller similarly
there is a bigger vector
yeah east artificial chromosome is a
bigger vector linear vector not circular
and it can carry bigger
dna stretch or gene of interest okay and
generally east artificial chromosome is
used for cloning bigger eukaryotic genes
which are obviously lengthier in size
so in plasmid vector what is the insert
size 5 to 25 kb okay the insert size is
clear that the insert size will be 5
kilo bases to 25 kilo bases at max
depending upon the different kinds of
plasmid vector the insert size varies
for example
p u c this is one example p u puc19
vector is a plasmid vector name plasmid
vector is a type and the example is
puc19 it has an insert size of 15
kilobases maximum okay kill 15 kilo base
pair max
now apart from the insert size this
plasmid vector as i said there is a
self-replicating component so it must
have
origin of replication known as re must
have that and also ah you know after we
insert the target dna
so we know that there are vectors and we
cut the vector at a particular part and
then we insert the target dna so for
that we need to have a cut site so we
need to have a restriction in the
nucleus cart site
okay which is known as
multiple cloning site
or poly linker because if this cloning
site is a place to cut the vector but
that place can be cut with multiple
restriction in the nucleus then we call
it multiple cloning site or we call it
poly linker region poly means again
multiple so poly linker multiple cloning
site is a site which can be cleaved by
multiple types of restriction indonesia
this can be equal r1 it can be damaged
one it can be hindi three different
examples are out there okay so a vector
must contain origin of replication the
restriction in the nucleus side
and obviously it has some insert size
and the last thing the vector must carry
is something
which helps us
to distinguish between
the vector
that is containing our target dna
and a vector without the target dna see
what happens is that let's say this is a
vector we cleave it with restriction
endonuclease vector is cleaved now our
target gna let's say this zigzag one is
inserted and there's a possibility that
we the vector can self-ligate without
inserting without having the target
dna
so there are these two kinds of plasmid
now this is a plasmid where you have a
target dna and the vector we call it
recombinant plasmid and there is a
plasmid we don't have any uh
target dna inserted so this is a normal
non-recombinant plasmid okay
so between recombinant plasmid and
non-recombinant plasmid how to dis
differentiate and distinguish to
distinguish that we need to have what is
known as selectable marker selectable
marker region okay so marker region of
the gene is a place which help us to
selectively choose
the recombinant plasmid from the
non-recombinant plasmid okay so what are
the components of the plasmid vector
here origin of replication known as ori
selectable marker which is most of the
case
resistance genes antibiotic
antibiotic resistant gene for example
tetracycline resistant gene
ampicillin resistant gene and so on
then there are multiple cloning sites
restriction digestion sites and
generally these multiple cloning sites
are present
within
the selectable marker region most of the
time and there are promoter region
if we are talking about a vector which
is uh
used as a expression vector then we need
a promoter region for a cloning vector
promoter region is not important but for
an expression vector the promoter region
is really important with which we can do
the transcription process
so this is one example let me erase all
this color here
it is all color
and let's see the pointer option okay so
what else we have we have origin of
replication this is the first point what
else we have we have uh the selectable
marker region now the selectable marker
antibiotic resistance uh genes can be
selectable marker antibiotic resistances
can be present apart from the selectable
marker both of them can be present
it must have a restriction sites
okay restriction site restriction
endonuclease known as polylinker region
or known as multiple cloning site or mcs
and sometimes it has promoter if the
plasmid vector is used as the expression
vector then only we need promoter
so these are all components of a plasmid
vector this is one example of a plasmic
vector known as puc19 you can see that
it has origin
it has a promoter what else it has it
has lag z and lag y gene as well
which is uh used uh along with the
multiple cloning site or mcs so mcs or
restriction endonucleoside is under
i mean within the lag z gene and apart
from that it also has this restriction
sites that is equal r1 side and also
ampicillin resistance site
which is selectable marker region that
is also for present so this is
selectable marker this is multiple
cloning site ah this is origin these are
the three
important component of any vector and
promoter is also there because
puc19 can be used both as a cloning
vector as a expression vector
now what are the advantages and
disadvantages of uh this plasmid vector
the advantage is that it's a small and
easy to handle this vector is very small
so it's very easy to handle easy to
purify
and the selection process and screening
process is less leversum easy to do and
useful for cloning very small dna
fragment if our target dna fragments or
target gene of interest is very small
then plasmids are the way to go because
plasmids are by default self-replicating
double-stranded circular dna and
maintenance of the plasmin inside of the
host cell is very easy because naturally
the bacteria maintains it and the copy
number is also huge 500 600 copy number
is very common like puc19 what is the
disadvantage the only disadvantage is
the insert size the insert size cannot
be more than
i can write this cannot be more than
15 kb i can write 15 kv not 10 15 kb
okay generally more than 10 kb length of
the sequence if you try to clone it if
you try to insert it and amplify it via
molecular cloning using a plasmid then
it causes uh you know problem it can
cause a problem in terms of
recombinant plasmid formation it can
cause it can possess problem in terms of
the maintenance of the plasmin inside
the host cell and the transformation
efficiency can be compromised rather
than that rest of the things are quite
easy quite
easy like a breeze that's why
plasmids are widely used molecular
cloning vehicles or molecular cloning
vectors that we use for the molecular
cloning process and expression in case
of expression as well okay that's all
about the plasmid vector and the example
of plasmid vector used in molecular
cloning two such example we'll talk
about we talk about pbr 322 and we will
also talk about the second example that
is p
u c
19 actually this p is a small ah letter
and the b are in caps similarly p
in puc19 in small uc in caps okay so
we'll talk about the pbr 322 and puc19
vector uh the details why they are named
like this and what they carry is there
any modification what are their
selective advantages and disadvantages
compared to each other and compared over
other vectors we'll talk all about this
in times to come
so if you like this video regarding the
plasmid vector please hit the like
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