pUC18: Plasmid, Cloning vector, Polylinker, [email protected]

Summary

In 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

• 🔬 The lecture is about the pUC18 cloning vector, a well-known plasmid used in genetic engineering.
• 🏫 The 'p' in pUC18 stands for plasmid, 'UC' stands for University of California, and '18' denotes its variant in the pUC series.
• 🧬 pUC18 is a cloning vector, not an expression vector, used for amplifying cloned genes in vivo.
• 📏 Ideal cloning vectors like pUC18 are small in size, allowing for the cloning of larger DNA fragments.
• 🚀 pUC18 includes a pLac promoter from the lac operon of E. coli, which can be induced by IPTG to initiate transcription.
• 🧩 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.
• 🔗 pUC18 has a multiple cloning site (MCS) with various restriction sites, enabling versatile DNA fragment cloning.
• 💊 The vector includes an ampicillin resistance gene, beta-lactamase, allowing for the selection of transformed cells.
• 🔄 The origin of replication (ori) in pUC18, derived from the pMB1 vector, ensures the plasmid can replicate independently within the host.
• 🔍 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 β-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

00:00

🧬 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.

05:01

🔬 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 β-D-1-thiogalactopyranoside), allowing for the transcription of genes. The presence of the lacZ' gene, a fragment of the β-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.

10:02

🛡️ 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 β-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.

15:16

🌟 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.

Keywords

💡Cloning Vector

A cloning vector is a DNA molecule used as a vehicle to carry foreign DNA fragments for various purposes such as replication, amplification, and expression. In the video, 'pUC18' is discussed as a well-known cloning vector, which is used to amplify the cloned gene of interest within a host cell, typically E. coli. The script mentions that cloning vectors are essential for downstream applications like subcloning or sequencing.

💡Plasmid

A plasmid is a small, circular DNA molecule found in bacteria and used in molecular biology as a cloning vector. The script describes plasmids as covalently closed, circular, extrachromosomal double-stranded DNA molecules. 'pUC18' is an example of an artificial plasmid used for cloning purposes.

💡Nomenclature

Nomenclature refers to the set of rules for naming things, especially in a scientific context. In the script, the nomenclature of 'pUC' is explained, where 'p' stands for plasmid, 'UC' stands for University of California, and the number '18' indicates a variant in the series of plasmids developed by the University.

💡Promoter

A promoter is a DNA sequence that acts as a starting point for the transcription of a gene into mRNA. In the video, the 'pLac' promoter is mentioned, which is isolated from the lac operon of E. coli and is used to initiate transcription when induced by IPTG (isopropyl β-D-1-thiogalactopyranoside).

💡Lac Operon

The lac operon is a set of genes in E. coli that are involved in the metabolism of lactose. The script explains that the 'pLac' promoter used in 'pUC18' is derived from this operon and is involved in the regulation of lactose metabolism when lactose is present and glucose is absent.

💡IPTG

IPTG is a non-metabolizable inducer of the lac operon, used in molecular biology to activate the expression of genes under the control of the lac promoter. The script describes how IPTG binds to the repressor of the lac operon, allowing RNA polymerase to bind to the 'pLac' promoter and initiate transcription.

💡Multiple Cloning Site (MCS) or Polylinker

The MCS or polylinker is a region in a cloning vector that contains multiple restriction enzyme recognition sites, allowing for the insertion of various DNA fragments. The script mentions that 'pUC18' has an MCS with ten different restriction sites, facilitating directional cloning and accommodating different DNA fragments.

💡Ampicillin Resistance Marker

An ampicillin resistance marker is a gene that confers resistance to the antibiotic ampicillin. In the script, it is mentioned that 'pUC18' carries such a marker, which is used for selecting transformed E. coli cells that have successfully taken up the plasmid.

💡Beta-Lactamase

Beta-lactamase is an enzyme that breaks down the beta-lactam ring of penicillin antibiotics, rendering them inactive. The script explains that the ampicillin resistance gene in 'pUC18' encodes beta-lactamase, which allows transformed E. coli to grow in the presence of ampicillin.

💡Origin of Replication (Ori)

The origin of replication is a specific DNA sequence where replication of the DNA molecule begins. In the script, it is mentioned that 'pUC18' contains an origin of replication that enables the plasmid to be replicated autonomously within the host cell, which is crucial for a high copy number of the plasmid.

💡Alpha Complementation

Alpha complementation is a method used to detect recombinants in cloning by using the alpha peptide encoded by the 'lacZ' gene. The script describes how in 'pUC18', the alpha peptide from the vector can associate with the omega peptide from the host to form a functional beta-galactosidase only if no insert is present, allowing for visual detection of recombinants and non-recombinants on X-gal containing media.

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

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primer

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and if you have any comment please let

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thank you all for listening see you

again with one more video until then bye

bye