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Plasmids

After restriction digestion, genes of interest are commonly inserted into plasmids , small pieces of typically circular, double-stranded DNA that replicate independently of the bacterial chromosome (see Unique Characteristics of Prokaryotic Cells ). In recombinant DNA technology, plasmids are often used as vectors , DNA molecules that carry DNA fragments from one organism to another. Plasmids used as vectors can be genetically engineered by researchers and scientific supply companies to have specialized properties, as illustrated by the commonly used plasmid vector pUC19 ( [link] ). Some plasmid vectors contain genes that confer antibiotic resistance ; these resistance genes allow researchers to easily find plasmid-containing colonies by plating them on media containing the corresponding antibiotic. The antibiotic kills all host cells that do not harbor the desired plasmid vector, but those that contain the vector are able to survive and grow.

Plasmid vectors used for cloning typically have a polylinker site , or multiple cloning site (MCS) . A polylinker site is a short sequence containing multiple unique restriction enzyme recognition sites that are used for inserting DNA into the plasmid after restriction digestion of both the DNA and the plasmid. Having these multiple restriction enzyme recognition sites within the polylinker site makes the plasmid vector versatile, so it can be used for many different cloning experiments involving different restriction enzymes.

This polylinker site is often found within a reporter gene , another gene sequence artificially engineered into the plasmid that encodes a protein that allows for visualization of DNA insertion. The reporter gene allows a researcher to distinguish host cells that contain recombinant plasmids with cloned DNA fragments from host cells that only contain the non-recombinant plasmid vector. The most common reporter gene used in plasmid vectors is the bacterial lacZ gene encoding beta-galactosidase, an enzyme that naturally degrades lactose but can also degrade a colorless synthetic analog X-gal , thereby producing blue colonies on X-gal–containing media. The lacZ reporter gene is disabled when the recombinant DNA is spliced into the plasmid. Because the LacZ protein is not produced when the gene is disabled, X-gal is not degraded and white colonies are produced, which can then be isolated. This blue-white screening method is described later and shown in [link] . In addition to these features, some plasmids come pre-digested and with an enzyme linked to the linearized plasmid to aid in ligation after the insertion of foreign DNA fragments.

The pC19 plasmid is 2686 bp long and is circular. There are a number of arrows of various lengths moving clockwise or counterclockwise around the circle. The circle has the number 1 at the very top and at about position 225 (moving clockwise from 1) is a plylinker site (MC) that contains the following restriction enzyme sites: HidIII, PSTI, SaII, XbaI, BamHI, SmaI, EcoRI. At the beginning of the polylinker site is lacZ-alpha. At position 2500 and moving counterclockwise is the amp gene.
The artificially constructed plasmid vector pUC19 is commonly used for cloning foreign DNA. Arrows indicate the directions in which the genes are transcribed. Note the polylinker site, containing multiple unique restriction enzyme recognition sites, found within the lacZ reporter gene. Also note the ampicillin ( amp ) resistance gene encoded on the plasmid.

Molecular cloning using transformation

The most commonly used mechanism for introducing engineered plasmids into a bacterial cell is transformation , a process in which bacteria take up free DNA from their surroundings. In nature, free DNA typically comes from other lysed bacterial cells; in the laboratory, free DNA in the form of recombinant plasmids is introduced to the cell’s surroundings.

Questions & Answers

how does Neisseria cause meningitis
Nyibol Reply
what is microbiologist
Muhammad Reply
what is errata
Muhammad
is the branch of biology that deals with the study of microorganisms.
Ntefuni Reply
What is microbiology
Mercy Reply
studies of microbes
Louisiaste
when we takee the specimen which lumbar,spin,
Ziyad Reply
How bacteria create energy to survive?
Muhamad Reply
Bacteria doesn't produce energy they are dependent upon their substrate in case of lack of nutrients they are able to make spores which helps them to sustain in harsh environments
_Adnan
But not all bacteria make spores, l mean Eukaryotic cells have Mitochondria which acts as powerhouse for them, since bacteria don't have it, what is the substitution for it?
Muhamad
they make spores
Louisiaste
what is sporadic nd endemic, epidemic
Aminu Reply
the significance of food webs for disease transmission
Abreham
food webs brings about an infection as an individual depends on number of diseased foods or carriers dully.
Mark
explain assimilatory nitrate reduction
Esinniobiwa Reply
Assimilatory nitrate reduction is a process that occurs in some microorganisms, such as bacteria and archaea, in which nitrate (NO3-) is reduced to nitrite (NO2-), and then further reduced to ammonia (NH3).
Elkana
This process is called assimilatory nitrate reduction because the nitrogen that is produced is incorporated in the cells of microorganisms where it can be used in the synthesis of amino acids and other nitrogen products
Elkana
Examples of thermophilic organisms
Shu Reply
Give Examples of thermophilic organisms
Shu
advantages of normal Flora to the host
Micheal Reply
Prevent foreign microbes to the host
Abubakar
they provide healthier benefits to their hosts
ayesha
They are friends to host only when Host immune system is strong and become enemies when the host immune system is weakened . very bad relationship!
Mark
what is cell
faisal Reply
cell is the smallest unit of life
Fauziya
cell is the smallest unit of life
Akanni
ok
Innocent
cell is the structural and functional unit of life
Hasan
is the fundamental units of Life
Musa
what are emergency diseases
Micheal Reply
There are nothing like emergency disease but there are some common medical emergency which can occur simultaneously like Bleeding,heart attack,Breathing difficulties,severe pain heart stock.Hope you will get my point .Have a nice day ❣️
_Adnan
define infection ,prevention and control
Innocent
I think infection prevention and control is the avoidance of all things we do that gives out break of infections and promotion of health practices that promote life
Lubega
Heyy Lubega hussein where are u from?
_Adnan
en français
Adama
which site have a normal flora
ESTHER Reply
Many sites of the body have it Skin Nasal cavity Oral cavity Gastro intestinal tract
Safaa
skin
Asiina
skin,Oral,Nasal,GIt
Sadik
How can Commensal can Bacteria change into pathogen?
Sadik
How can Commensal Bacteria change into pathogen?
Sadik
all
Tesfaye
by fussion
Asiina
what are the advantages of normal Flora to the host
Micheal
what are the ways of control and prevention of nosocomial infection in the hospital
Micheal
what is inflammation
Shelly Reply
part of a tissue or an organ being wounded or bruised.
Wilfred
what term is used to name and classify microorganisms?
Micheal Reply
Binomial nomenclature
adeolu

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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