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A diagram explaining molecular cloning. Both foreign DNa and a plasmid are cut with the same restriction enzyme. The restriction site occurs only once in the plasmid in the middle of a gene for and enzyme (lacZ). The plasmid also contains an ampicillin resistang gene. The restriction enzyme leaves complementary sticky ends on the foreign DNA fragment and the plasmid. This allows the foreign DNA to be inserted into the plasmid when the sticky ends anneal. Adding DNA ligase reattaches the DNA backbones. These are recombinant plasmids. The plasmids are combined with a culture of living bacteria. Many of the bacteria do not take any plasmids into their cells. Many take plasmids that do not have the foreign DNA in them and a few take up the recombinant plasmid. The bacteria that take up the recombinant plasmid cannot make the enzyme from the gene that the fragment was inserted into (lacZ). They also carry a gene for resistance to the antibiotic ampicillin, which was on the original plasmid. To find the bacteria with the recombinant plasmid, the bacteria are grown on a plate with the antibiotic ampicillin and a substance that changes color when exposed to the enzyme produced by the lacZ gene. The ampicillin will kill any bacteria that did not take up a plasmid. The color of the substance will nto change when the gene for lacZ contains the foreign DNA insert. These are the bacteria with the recombinant plasmid that we want to grow.
The steps involved in molecular cloning using bacterial transformation are outlined in this graphic flowchart.
  • What is the original function of a restriction enzyme?
  • What two processes are exploited to get recombinant DNA into a bacterial host cell?
  • Distinguish the uses of an antibiotic resistance gene and a reporter gene in a plasmid vector.

Creating a genomic library

Molecular cloning may also be used to generate a genomic library . The library is a complete (or nearly complete) copy of an organism’s genome contained as recombinant DNA plasmids engineered into unique clones of bacteria. Having such a library allows a researcher to create large quantities of each fragment by growing the bacterial host for that fragment. These fragments can be used to determine the sequence of the DNA and the function of any genes present.

One method for generating a genomic library is to ligate individual restriction enzyme-digested genomic fragments into plasmid vectors cut with the same restriction enzyme ( [link] ). After transformation into a bacterial host, each transformed bacterial cell takes up a single recombinant plasmid and grows into a colony of cells. All of the cells in this colony are identical clones and carry the same recombinant plasmid. The resulting library is a collection of colonies, each of which contains a fragment of the original organism’s genome, that are each separate and distinct and can each be used for further study. This makes it possible for researchers to screen these different clones to discover the one containing a gene of interest from the original organism’s genome.

A diagram showing the generation of a genomic library. The diagram begins with DNA being extracted from the organism (in this case a worm) and cut into fragments. The DNA fragments are then each inserted into a different plasmid. This produces many fragments each with a different insert from the genome. Bacteria are then transformed with these vectors. Each bacterium replicates producing colonies of clones each containing a single DNA fragment from the original organism.
The generation of a genomic library facilitates the discovery of the genomic DNA fragment that contains a gene of interest. (credit “micrograph”: modification of work by National Institutes of Health)

To construct a genomic library using larger fragments of genomic DNA, an E. coli bacteriophage, such as lambda , can be used as a host ( [link] ). Genomic DNA can be sheared or enzymatically digested and ligated into a pre-digested bacteriophage lambda DNA vector. Then, these recombinant phage DNA molecules can be packaged into phage particles and used to infect E. coli host cells on a plate. During infection within each cell, each recombinant phage will make many copies of itself and lyse the E. coli lawn, forming a plaque. Thus, each plaque from a phage library represents a unique recombinant phage containing a distinct genomic DNA fragment. Plaques can then be screened further to look for genes of interest. One advantage to producing a library using phages instead of plasmids is that a phage particle holds a much larger insert of foreign DNA compared with a plasmid vector, thus requiring a much smaller number of cultures to fully represent the entire genome of the original organism.

A diagram showing the production of a phage library. The cellular genome and phage genome are digested with the same restriction enzyme. The cellular DNA fragments are built into recombinant phage particles (each particle contains a different fragment of cellular DNA). E. coli is then infected with the recombinant phages. Each plaque in the bacterial lawn contains phages with a unique fragment from the original genome.
Recombinant phage DNA molecules are made by ligating digested phage particles with fragmented genomic DNA molecules. These recombinant phage DNA molecules are packaged into phage particles and allowed to infect a bacterial lawn. Each plaque represents a unique recombinant DNA molecule that can be further screened for genes of interest.

Questions & Answers

I am about to enter school and am going for MCB
Olugbenga Reply
What is DNA damage and repair
Sree Reply
removal of nucleotide bases from DNA is damage n addition or insertion of bases in DNA is repair.
Rashmi
replication is a term related to generation of new DNA from a parental DNA.
Rashmi
role of microorganism as pathogens
Parteek Reply
to live inside the host body, reproduction n destroy the immune system of host body.
Rashmi
what is replication
rajesh
please give me ans
rajesh
replication is a term related to generation of new DNA from parental DNA.
Rashmi
difference between endotoxin and exotoxin
Binkheir Reply
toxins released inside the cell or a body is endotoxin n toxins released outside the cell or a body is exotoxin.
Rashmi
please I want the names of bacteria and the diseases they cause.
Ibrahim Reply
there are many we classify it according to their shapes gram negative or gram positive
Tharra
so u may refer the book u guess or by when u want I can do a list for you and send u here
Tharra
😊
Tharra
please do a list for me
Ibrahim
I have the lost but it's too much to type ..
Tharra
MSc entrance prepare books and questions LA etha base pani irukum..
Guhan Reply
kushal always prescott is the imp book to follow.search some objective mcqs books based on your syllabus
deepthi Reply
I want to clear entrance of ms university so I have no more idea so my preparation is based on my bachlor studies.
Kushal
what are the opportunistic infection in aids stage
Kalkak
certain cancer or pneumonia 🤔
Kushal
penicillium crysogenum
Kushal Reply
salam am also studying microbiology in university of Ilorin
Abubakar Reply
Salam am studying physiotherapy in bayaro university kano
Tasiu
Walaikum salam
Hakeeb
Hw u
Tasiu
HRU frinds
Arhaan
any good reference for oncology?!!!!
Pankaj Reply
K.D tripathi
Vinod
what is the microbiology
Fadumo Reply
it is the study of microorganisms
Sneha
microorganis means
Ghuge
the organism can not be seen by eyes they can be seen with the help of microscope
rajesh
microbiology is the study of microorganism and microorganism are such as bacteria ,parasite, viruses and fungi and etc.. those are cant be seen with asked eyes and can be seen with help of microscope . I hope this helps you
Tharra
what is virology
Bismarck Reply
the study of viruses
Adan
what is myrology
Bismarck
myrology or mycology?
Adan
mycology
Bismarck
the study of fungi.
Adan
Mycology study fungi
Hakeeb
study of viruses
Rashmi
what are the major topics that you cover under micro biology
Bismarck Reply
virology,mycology,bacteriology
Adan
identification n application of microorganisms
Rashmi
How do u apply micro biology in medicine
Bismarck Reply
in preventing, diagnosis and treatment of infectious diseases.
Adan
and also in carrying out test And Analysis
Hadiyat
what is micro biology
Bismarck Reply
study of microorganisms
Adan
thanks
Bismarck
OK thanks alot
Bismarck

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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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