Plasmid DNA vector in gene cloning | plasmid vector | pbr322 vector | puc 19 vector

Summary

This 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

00:00

🧬 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.

05:00

🔬 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.

10:00

📈 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.

Keywords

💡Plasmid Vector

A plasmid vector is a small, circular, self-replicating DNA molecule that is used in molecular biology as a vehicle for gene delivery and amplification. It is a fundamental component in recombinant DNA technology. In the video, plasmid vectors are discussed as essential tools for cloning, where they carry the target gene into a host cell, such as E. coli, for replication and study.

💡Recombinant DNA Technology

Recombinant DNA technology refers to the manipulation of DNA molecules to create new combinations of genetic material. It is central to genetic engineering and biotechnology. The script discusses this technology in the context of using plasmid vectors to insert and amplify target genes, highlighting its importance in modern molecular biology.

💡Cloning Vector

A cloning vector, as mentioned in the script, is a tool used to deliver a target gene into a host cell where it can be amplified. Plasmid vectors are a type of cloning vector that can replicate independently of the host cell's chromosomal DNA, allowing for the production of many copies of the inserted gene.

💡Expression Vector

An expression vector is a specific type of cloning vector that not only carries the target gene into a host cell but also facilitates its transcription and translation into a protein. The script explains that while plasmid vectors can serve as cloning vectors, they can also be engineered to function as expression vectors when a promoter region is included.

💡Self-replicating

Self-replicating refers to the ability of a molecule or entity to make copies of itself. In the context of the video, plasmids are described as self-replicating DNA molecules, which is a key feature that allows them to be used as vectors in cloning. This property ensures that once inside the host cell, the plasmid, along with the inserted gene, can be amplified.

💡Insert Size

Insert size refers to the amount of DNA that can be accommodated within a vector. The script specifies that plasmid vectors typically have an insert size limitation, ranging from 5 to 25 kilobases, depending on the vector. This is an important consideration when choosing a vector for cloning, as the size of the target gene must fit within this range.

💡Selectable Marker

A selectable marker is a gene that provides a phenotype which can be selected for, allowing for the identification of successfully transformed cells. In the script, it is mentioned that plasmid vectors contain selectable markers, often antibiotic resistance genes, which help distinguish recombinant plasmids carrying the target gene from non-recombinant ones.

💡Multiple Cloning Site (MCS)

A multiple cloning site, or polylinker, is a region within a vector that contains a variety of recognition sites for different restriction enzymes. As discussed in the script, this feature allows for the insertion of the target DNA at various points, providing flexibility in cloning experiments.

💡Origin of Replication (ori)

The origin of replication, or ori, is a specific sequence within a DNA molecule that serves as the starting point for DNA replication. The script explains that plasmid vectors must contain an ori to enable them to replicate within the host cell, which is crucial for the cloning process.

💡Copy Number

Copy number refers to the number of copies of a plasmid that can exist within a single host cell. The script mentions that the copy number can vary, with some plasmids having the capacity to be present in hundreds or even thousands of copies per cell, which influences the efficiency of gene amplification.

💡PBR322 and pUC19

PBR322 and pUC19 are specific examples of plasmid vectors mentioned in the script. They are named according to the characteristics they carry, such as antibiotic resistance and the size of the insert they can accommodate. The script promises to delve into the details of these vectors, including their advantages and disadvantages, in subsequent discussions.

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

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thank you bye