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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

what is the size of virus
Beatrice Reply
What is the difference between TVC and Bioburden test
Mohamed
?
Mohamed
structure of bacteria and 10 types
Jennifer Reply
what is accidental host?
Domingo Reply
what is endomembrane system
Ikpi Reply
what is human anatomy
Ikpi
okay. Go ahead and ask
Blessing Reply
Industrial microbiology mcq
mohamed
Okay. What's your question?
Blessing
life arises from living matter or live organism.
Swami Reply
yes
sildra
I think live matter arises from non living matter
sildra
I dont think so...can u explain with an example
Manya
living maters made by non living matters
sildra
non living matters like stones? rocks?
Manya
eno
sildra
no
sildra
then?
Manya
cells are made by C N O minerals etc
sildra
I mentioned these as non living maters
sildra
that's all
sildra
cells are made up of those things but they originate from living things..
Manya
ok
sildra
Ok..good chat:-)
Manya
where are you from
sildra
thanks
sildra
India...u?
Manya
Tamil nadu
sildra
I am from Maharashtra
Manya
what about your studies
sildra
completed bsc.. preparing for msc entrance...wbu?
Manya
same
sildra
are you microbiologist
sildra
yes i am
Manya
good
sildra
what s the scope for micro in ur state?
Manya
did you find your college to higher studies
sildra
have to give an entrance exam for every college here...so lets c
Manya
food industries, medical lab, vaccine industries ,etc
sildra
hoping for pune University...wbu?
Manya
great!
Manya
is that centeral University right
sildra
what is your namr
sildra
hello
Udhaya
Hi
Maruf
Family kindly help me with this question? 1) Shortlist the configurative measurements of the following human anatomical ranges of÷ - Blood ( haemeglobin) in both male and female - Haematocytes in both male and female - Hepatocytes in both male and female - Lymphocyte / T. Lymphocytes in both male
Gifted
My names are Gift Mwale and am a Zambian. Kindly help me with this research which goes like this... 1) Shortlist the configurative measurements of the following human anatomical ranges of ÷ - Blood ( haemeglobin) in both male and female - Hepatocytes in both male and female - Haematocytes in both
Gifted
please what is the full meaning for TCDS
UDEME
from a single cell
Freedom
tcds means transcranial direct current stimulation...in this small electric currents are given to brain( specific parts) to help increase brain performance or to help with depression.. current should be in range 0.5-2.0mA
Manya
what's underlying disease relating unsanitary diet microorganism with the highest rate of epidemology solution and efficacy leading molecules elucidated structural solutions
feven
please can anybody talk about brain tumour and its cure.
BELLO Reply
enlargement of the thyroid gland resulting in over production of hormone.
Kamal Reply
What can u say on Thyroid Cancer?
Abdulkareem
Please, talk about the thyroid cancer.
BELLO
explain the Grave's disease
John Reply
what is cell
Avi Reply
is unit of life
Kamaluddeen
Ok
mohamed
who is an industrial microbiologist
Cynthia Reply
I want to know the biochemical composition of bacteria
Josh Reply
It contains peptidoglcon, DNA nd RNA
Asiya
what are Carrier protein
Ikpi
bacteriophage disadvantage
Momina Reply
disease due to __________ abnormalities are termed primary immunodeficiencies
Tayee Reply
Some primary immunodeficiencies are due to a defect of a single cellular or humoral component of the immune system.
Prince
Examples of primary immunodeficiencies include: chronic granulomatous disease, X-linked agammaglobulinemia, selective IgA deficiency etc
Prince
thank you
Nana
explain microbial mutation
Emerald
what is mutation
Cynthia Reply
alteration in genetic makeup
UDEME

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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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