Basics of Molecular Cloning (1)

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Promega Corporation
Education Resources
Unit 006
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Defining Cloning
“Cloning” is a loaded term that can be used to mean very different things.
 Cutting a piece of DNA from one organism and inserting it into a vector where it can be replicated by a host organism. (Sometimes called subcloning, because only part of the organism’s DNA is being cloned.)
Using nuclear DNA from one organism to create a second organism with the same nuclear DNA
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Restriction Enzymes
	Restriction Enzymes (also called Restriction Endonucleases) are proteins that cleave DNA molecules at specific sites, producing discrete fragments of DNA.
	Restriction Enzymes (RE) were first isolated by Nathans and Smith in 1970.
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Why Restriction Enzymes?
Why would bacterial cells contain proteins that cleave DNA at specific sequences?
Generally restriction enzymes are thought to protect bacterial cells from phage (bacterial virus) infection. Bacterial cells that contain restriction enzymes can “cut up” invasive viral DNA without damaging their own DNA.
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Joining DNA Fragments
In 1972, Paul Berg and colleagues made the first “artificial” recombinant DNA molecule.
Demonstrated that the DNA of Simian virus 40 could be linearized by EcoR1
Created a circular DIMER of Simian virus DNA by joining two linearized fragments
Also inserted pieces of Lambda phage DNA into linearized Simian 40 virus molecule.
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Isolating Genes	
Herbert Boyer and Stanley Cohen built on the work of Berg, Nathans and Smith to use restriction enzymes to isolate a single gene, place it into a plasmid vector.
Bacterial cells were then transformed with the recombinant plasmid.
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The bacteria host cells replicated the plasmid, producing many copies of the gene, thus amplifying it.
The practical application was that expensive human protein products, like insulin, which were used to treat disease, could eventually be produced from recombinant molecules in the laboratory using bacteria or another host.
Human protein products like insulin could be used in very large quantities from the recombinant molecule. Patients no longer had to use insulin isolated from pigs or cows.
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Plasmid Vectors
Plasmids are circular pieces of DNA found naturally in bacteria.
Plasmids can carry antibiotic resistance genes, genes for receptors, toxins or other proteins.
Plasmids replicate separately from the genome of the organism.
Plasmids can be engineered to be useful cloning vectors.
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Plasmid Vectors (continued)
Plasmid vectors can be designed with a variety of features:
Antibiotic resistance
Colorimetric “markers”
Strong or weak promoters for driving expression of a protein
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Antibiotic Resistance Markers
Antibiotic 
Resistance 
Gene
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Multiple Cloning Region
Multiple
Cloning 
Region
The cloning marker for this plasmid is the lacZ gene.
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Cloning a Piece of DNA
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AvaI
Cut plasmid vector
with AvaI
AvaI
AvaI
5´
3´
Excise DNA insert of interest from source using Ava I
Ligate the insert of interest
 into the cut plasmid
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Performing the Restriction Digests
You will need to set up a restriction digest of your plasmid vector and your DNA of interest
Restriction enzymes all have specific conditions under which they work best. Some of the conditions that must be considered when performing restriction digest are: temperature, salt concentration, and the purity of the DNA
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Purify your DNA Fragments
The insert of interest that you want to clone into your plasmid needs to be separated from the other DNA
You can separate your fragment using Gel Electrophoresis
You can purify the DNA from the gel by cutting the band out of the gel and then using a variety of techniques to separate the DNA from the gel matrix
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Ligation
Ligation is the process of joining two pieces of DNA from different sources together through the formation of a covalent bond.
DNA ligase is the enzyme used to catalyze this reaction.
DNA ligation requires ATP.
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Transforming Bacteria
After you create your new plasmid construct that contains your insert of interest , you will need to insert it into a bacterial host cell so that it can be replicated.
The process of introducing the foreign DNA into the bacterial cell is called transformation.
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Competent Host Cells
Not every bacterial cell is able to take up plasmid DNA.
Bacterial cells that can take up DNA from the environment are said to be competent.
Can treat cells (electrical current/divalent cations) to increase the likelihood that DNA will be taken up
Two methods for transforming: heat shock and electroporation
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Selecting for Transformants
The transformed bacteria cells are grown on selective media (containing antibiotic) to select for cells that took up plasmid.
For blue/white selection to determine if the plasmid contains an insert, the transformants are grown on plates containing X-Gal and IPTG. (See notes for slide 11.)
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What did the cells take up?
Plasmid only
Plasmid with insert cloned
Foreign DNA from the environment
Nothing
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Expressing your cloned gene
Even if your plasmid contains insert, it may not be able to generate functional protein from your cloned DNA.
The gene may not be intact, or mutations could have been introduced that disrupt it.
The protein encoded by the gene may require post-translational modifications (i.e., glycosylation or cleavage) to function.
Also, some enzymes are a complex of peptides expressed from separate genes.
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Expressing your cloned gene
Expression of a cloned gene can be accomplished by:
The E. coli host
Mammalian cells (if the plasmid used is designed for expression in mammalian cells)
Using an in vitro using a cell-free system. (See education resources Unit 001: The relationship between genes and proteins)
Restriction Enzymes are named for the bacterial species and strain from which they are derived. 
For instance EcoRI is isolated from E. coli R strain and it is the first RE named from that strain. 
BamHI is isolated from Bacillus amyloliquefaciens, strain H, and it is the first RE named from that strain.
Conventionally, the abbreviation of the species name is written with the first letter capitalized, and the abbreviation italicized. The strain designation, when provided, is given as a uppercase letter, and the number is a Roman numeral (EcoRI). While many sources are following this convention, many others are eliminating the italics from the RE name (EcoRI). Check the instructions to authors or appropriate style guide to determine the correct formatting for any document you are creating.
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The this landmark work to use restriction endonucleases, DNA polymerase and ligase to create recombinant DNA molecules is published in two papers in the Proceedings of the National Academy of Sciences.
Morrow, J.F. and Berg, P. (1972) Cleavage of Simian virus 40 DNA at a unique site by a bacterial restriction enzyme. Proc. Natl. Acad. Sci. USA. 69, 3365–9. The full text of this article is available here: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=4343967 
Jackson, D.A., Symons, R.H. and Berg, P. (1972) Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc. Natl. Acad. Sci. USA. 69, 2904–9. The full text of this article is available here: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=4342968
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A short synopsis of the meeting of Boyer and Cohen is available here: http://web.mit.edu/invent/a-winners/a-boyercohen.html 
This work was published in 1973: 
Cohen, S.N., Chang, A.C.Y., Boyer, H.W. and Helling, R.B. (1973) Construction of biologically functional bacterial plasmids in vitro. 70, 3240–4.
A PDF of the entire paper can be accessed
here: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=4594039 
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More information about Boyer and Cohen’s work:
http://www.genomenewsnetwork.org/resources/timeline/1973_Boyer.php
An interesting perspective on the patent issue and the beginnings of the relationship of universities with industry in molecular biology: 
http://www.ias.ac.in/currsci/mar252009/760.pdf
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A plasmid is a closed, circular piece of DNA that is found outside of the genome of the bacterial cell; sometimes referred to as “extra chromosomal DNA”. The plasmid requires an origin of replication (ORI) before it can be replicated by the bacterial host cell.
The piece of DNA that is cloned into a plasmid vector is referred to as the “insert”.
Plasmids can be high- or low-copy number. High-copy number plasmids are replicated frequently by the bacterial host, creating many copies and thereby “amplifying” the cloned insert many fold. Low-copy number plasmids are replicated more slowly, so the bacterial host cells do not contain very many copies of the plasmid. Low-copy number plasmids are sometimes used for cloning pieces of DNA that encode proteins that are toxic to the host cell if they are expressed at high levels.
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To determine whether your bacterial host cells have “taken up” your plasmid when you are cloning, most plasmids contain some sort of marker for antibiotic resistance. In this instance, the pGEM-3Z Vector contains a gene for ampicillin resistance (Ampr). Bacterial cells that contain this plasmid will be resistant to the antibiotic ampicillin. Bacterial cells that do not contain this plasmid (presuming they contain no other source of antibiotic resistance) will be sensitive to the antibiotic amplicillin. This difference allows you to “transform” your cells with the plasmid and then grow the cells in the presence of antibiotic. Only those cells that have “taken up” the plasmid should grow.
Kanamycin and Tetracycline are two other antibiotics that are commonly used to select for the presence of a plasmid in bacterial host cells.
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Most commercially designed plasmid vectors contain a multiple cloning region for inserting your DNA of interest. This region is an area of the plasmid vector that contains many common restriction enzyme sites. The advantage of a multiple cloning region is that you have a choice of restriction enzyme sites to use when you excise your DNA of interest from the genome of the organism from which it is obtained to put it in the plasmid vector.
In many plasmids the multiple cloning region disrupts some sort of marker gene. For the plasmid shown above, the marker gene is lac Z (the beta-galactosidase gene). If your vector and E. coli strain are compatible with blue/white screening, which takes advantage of intracistronic α-complementation to regenerate β-galactosidase activity, you can use blue/white screening to select for the presence of inserted DNA in your vector.
Many E. coli strains used for cloning and propagation of plasmids contain a chromosomal deletion of the lac operon but carry an F´ episome that provides the remaining coding sequence of the lacZ gene. The functional lacZ gene product, β-galactosidase, is produced when the lacZ coding information missing on the F´ episome is provided by the information contained in the plasmid. This activity is detected by plating bacteria transformed by plasmids on plates containing isopropyl β-D-thiogalactopyranoside (IPTG; an inducer of the lac promoter) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal; a dye that produces a blue color when hydrolyzed by β-galactosidase). When the reading frame of the α peptide is disrupted by insertion of a foreign DNA fragment or deletion of vector sequences, α-complementation does not occur, and the bacterial colonies remain white or occasionally light blue.
So, if you successfully insert your DNA of interest into the plasmid using one of the restriction enzyme sites in the multiple cloning region, the lacZ gene no longer supplies the necessary information to produce functional beta-galactosidase. Therefore bacterial cells that contain plasmid with insert will not be able to hydrolyze X-gal substrate, and they will produce white (or occasionally light blue) colonies.
If your DNA of interest is not successfully cloned into the plasmid, the plasmid does provide the information the bacterial host cell needs to produce beta-galactosidase, and the cells will be able to hydrolyze the X-gal substrate. They will produce dark blue colonies.
So, most cloning plasmids will contain two markers:
An antibiotic marker that will allow only those bacteria with plasmids to grow on medium containing the appropriate antibiotic, and
A cloning marker, such as lacZ, that will allow you to distinguish plasmids with insert from plasmids without insert.
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For instance if you are cloning a piece of DNA that is flanked on the 5´ and 3´ ends by an AvaI site, you could excise it from the source using AvaI. This would give you a fragment with overhangs at the 5´ and 3´ sites that you could then insert into the Ava I site of the plasmid shown here.
If the original size of the uncut plasmid was 3.0kb, and you add a 0.75kb insert, you will be able to verify the addition of the insert by analyzing uncut plasmid, cut plasmid, uncut plasmid with insert, and cut plasmid with insert on an agarose gel.
Alternatively, if you are cloning a piece of DNA that is flanked at the 5´ end by an XbaI site and at the 3´ end by a KpnI site, you could cut out your DNA of interest using those two enzymes, digest the plasmid vector with the same to enzymes and clone your DNA of interest between those to RE sites of the multiple cloning region. The advantage of this strategy is that your insert will be cloned directionally (i.e., it will be inserted with the 5´ end at the XbaI site of the vector and the 3´ end at the KpnI site). You will be able to predict the orientation of your insert within the vector. (Teaching suggestion: have your students sketch what such a cloning strategy would look like.)
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The Restriction Enzymes Resource on the Promega Website describes in detail the factors that must be considered when performing a restriction digest and gives several examples of typical restriction enzyme reactions. This guide is available at: http://www.promega.com/guides/re_guide/toc.htm 
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Promega offers a variety of systems for isolating DNA from agarose gels. These systems can be found here: http://www.promega.com/applications/dna_rna/fragment.htm 
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There are two primary methods for transforming bacterial cells: heat shock and electroporation. In both cases, the bacterial cells have to be made competent or permeable to plasmids that you would like the cell to propagate. To create competent cells for either transformation method used, bacterial cells are grown to logarithmic phase and harvested. Cells growing exponentially can be rendered competent more easily than cells at other stages of growth. After harvesting, the cells are treated differently. Chemically competent cells are created using a series of cold salt washes to disrupt the cell membranes, preparing the cells to accept plasmid DNA (1,2). For electrocompetent cells, the cells are chilled and washed with cold deionized water and 10% glycerol (3,4). A low-salt environment is important when electrical currents are involved.
To introduce the desired plasmid into chemically competent cells, the plasmid DNA is mixed with chilled cells and incubated on ice to allow the plasmid to come into close contact with the cells. The plasmid-cell mixture then is briefly heated to 45–50°C, allowing the DNA to enter the cell through the disrupted membrane. The heated mixture is then placed back on ice to retain the plasmids inside the bacteria. Many cells do not survive the rapid temperature change but enough maintain integrity to keep the plasmid and, when medium is added, recover and divide.
For electroporation, the
competent cells also sit on ice with the plasmid DNA. However, the plasmid-cell mixture is exposed to an electrical current, opening pores in the cell membrane so that the plasmid can enter the cell. Some cells do not survive this treatment but many are able to replicate once medium is added. If the plasmid DNA solution has too much salt in it, arcing can occur, compromising the transformation.
Depending on the transformation method used, a plasmid can enter the cell through holes or pores in the bacterial cell wall created by salt washes and heat treatment or no-salt washes and electroporation. Both methods allow efficient recovery of transformed cells using antibiotic selection for the plasmid of interest.
1. Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–80. 
2. Hanahan, D. (1985) In: DNA Cloning: A Practical Approach Vol. 1, Glover, D. M. ed., IRL Press, Oxford, p. 109–35. 
3. Calvin, N.M. and Hanawalt, P.C. (1988) High-efficiency transformation of bacterial cells by electroporation. J. Bacteriol. 170, 2796–801. 
4. Seidman, C.E. et al. (1994) In: Current Protocols in Molecular Biology, Ausebel, F.M. et al. John Wiley & Sons, Inc., New York, Unit 1.8.
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The markers on the plasmid will help you select bacterial colonies that contain the plasmid into which you have successfully cloned your DNA of interest.
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