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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

characteristic of Gram negative bacteria
jane Reply
Characteristics of Gram Negative Bacteria As with Gram positive bacteria, Gram negative bacteria also contain the peptidoglycan polymer in their cell wall. While this polymer is thin (2 to 4 nanometers in thickness with just about 3 layers of peptidoglycan) in Gram negative bacteria, it's also com
Kaviya
it's also composed of long glycan strands that are cross-linked by peptide molecules. This composition serves a number of functions including protecting the bacterial cell from lysis
Kaviya
Good shot
Enoch
Thanks 😊
Kaviya
what was Hans Christian Gram's supported in the modern Microbiology?
Wilson Reply
what is microbial growth
Chisa Reply
The organism responsible for vulva ulcers
nyiter Reply
Why are vascular pathogen poorly communicable from person to person?
Aj Reply
Most vascular pathogens are poorly communicable from person to person because they need a medium to be communicated i,e a vector that would carry them from one person to other
Kaviya
what's the habit of protista
Afieahngwi Reply
They show both autotrophic and heterotrophic mechanisms...
Swetha
thanks
Afieahngwi
welcome...
Swetha
let me mention some water. Air .Food and so on
Gattiek Reply
causes of infectious diseases
Afieahngwi Reply
water.Air
Gattiek
infectious disease are caused by pathogenic micro organisms like bacteria ,fungi..
Swetha
What is pasteurization?
Wilson
are fungi prokaryote or eukaryotes?
Afieahngwi Reply
fungi are eukaryotes.
Swetha
All fungi are eukaryotes. Even micro fungi.
Lad
have..complex cellular organization and membrane bound nucleus ...and..also... having loops of DNA( like plasmids) as.bacteria
Swetha
what enzyme replaces rna nucleotides with dna nucleotides during replication?
Remi Reply
an enzyme called DNA ligase.
Jael
describe the acid fast staining procedure used in the diagnosis of tuberculosis
Salma Reply
bacterial morphology
lf_ Reply
what is the difference between biogenesis & abiogenesis
Mayuri Reply
biogenesis is when living comes out from other living things as a result of reproduction while a biogenesis is the process where living things comes out from non living things
Usman
living things come form other form living things is biogenesis. ....right?
Mayuri
what is mean by pasturation method?
Mayuri Reply
Pasteurization is a process that kills harmful bacteria and creates an extended shelf life for your milk. ... It's pretty simple—we take the milk from the cows, we rapidly heat it to a high enough temperature to kill the bacteria, and then we cool it back down before packaging and shipping it to you
Kaviya
tell me about abiogenessis &biogenesis
Mayuri
discribe aristol spontaneous generation theory in brif
Mayuri Reply
The Greek philosopher Aristotle (384–322 BC) was one of the earliest recorded scholars to articulate the theory of spontaneous generation, the notion that life can arise from nonliving matter. Aristotle proposed that life arose from nonliving material if the material contained pneuma (“vital heat”).
Kaviya
thank you 😊
Mayuri
No mention dear 😊
Kaviya

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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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