Why does the DNA need to be in the form of a plasmid when pasted in a bacteria and not just the string of DNA cut by the restriction enzyme?

There are several reasons: 1) Linear DNA is unstable because there are enzymes present within all organisms (including bacteria) that degrade linear DNA molecules. 2) Vectors contain a sequence (known as the origin of replication) that causes the DNA to be replicated within the bacteria — this is necessary to maintain at least one copy of the new DNA per bacterium as the cells divide. 3) Vectors usually contain at least one sequence that allows selection for the vector (e.g. antibiotic resistance) — this is discussed in this article. 4) Vectors can also be used to do different things with the DNA. A common example of this would be an expression vector — this causes the DNA to be transcribed and translated and would allow you to examine the protein encoded in the cloned DNA. Does that help?

When the bacteria taking up the plasmid. How can we be sure that the bacteria used aren’t having any plasmid in it? To make sure that when we grow it on agar the bacteria got the recombinant DNA. What if. The original bacteria we use to take up the recombinant plasmids are already having its own plasmid. So how can we differentiate them then?

Good question :-) It could be difficult to know if you were just using a random bacteria isolated from nature — especially since there are likely to be many thousands of different plasmids (1730 were present in a sequence database as of 2009). We could sequence all the DNA inside the bacteria, but that is still a lot of work ... However that doesn't matter as much as you might think. For example, assume we are using a plasmid that contains a marker (selectable gene) encoding resistance to ampicillin. All we need to know is that the bacteria were are transforming are not already resistant to ampicillin. This is easy to test — we just try growing the bacteria in the presence of ampicillin, if they don't then we can use our plasmid. In practice microbiologists have domesticated strains of bacteria (a favorite is _Escherichia coli_ — often abbreviated to _E. coli_) that have been studied for decades. In almost all cases you would be using one of these well characterized strains and so would not need to worry about whether there were unknown plasmids.

Are identical twins also clones ?

The short answer is no. While they share a lot of DNA, there are also mutations that naturally occur within your cells, so no two people can share exactly the same DNA.

Do the bacteria ever make mistakes in the replication process? If not, why? If they do, how can we still call Dolly the sheep a clone if the original sheep is actually slightly different?

This is DNA cloning, not the actual cloning of organisms. For more information on cloning, visit this webpage: https://en.m.wikipedia.org/wiki/Cloning. Hope you find it useful! :)

How is the cutting and joining of DNA monitored?

To assess whether a "digest" (restriction enzyme cutting reaction) is complete, we usually run a small sample of the digest on an agarose gel§ with a "ladder" sample containing fragments of known sizes and a small sample of uncut DNA. This comparison allows us to see whether we got fragments of the expected sizes and how much uncut plasmid still remains. We generally don't directly check whether a ligation has worked — ligation is very reliable and it is usually easier to just transform the DNA into a new host bacteria. We then purify plasmid DNA and use restriction digests, PCR †, or sequencing to test whether we got the desired outcome. §For details see: https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/v/gel-electrophoresis-dna †For details see: https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/v/the-polymerase-chain-reaction-pcr

How can we clone a gene that is unknown sequence ?

You can use partial digestion method and expression in bacterial vectors and finally antibiotics to select your cloned vectors. Look at this: https://www.researchgate.net/post/How_do_i_clone_genes_with_unknown_sequence particularly this paper has protocol: https://www.researchgate.net/publication/10637344_Partial_characterization_of_a_transposon_containing_the_tetA_determinant_in_a_clinical_isolate_of_Acinetobacter_baumannii using tetA resistance gene as a marker on a plasmid (resistance to Tetracycline).

Why bacterias without plasmids will die?What are the functions of plasmid?

*Plasmids* are usually present in bacteria, and plasmids can replicate its own DNA independently of the bacteria, which is why it is often used in DNA cloning. Plasmids usually have an *antibiotic resistant gene*, so the bacteria won't die in the antibiotic. For more information, visit this webpage: https://en.m.wikipedia.org/wiki/Plasmid. I hope this answers your question! :)

Could you have a vector other than the bacterial plasmid for instance a bacteriophage

Yes, though every case I know of involves a phage based plasmid (known as a "phagemid") that is manipulated as a bacterial vector before being converted into a bacteriophage. I haven't personally used phagemids and suspect they are no longer commonly used, but you can learn more about this technology here: https://en.wikipedia.org/wiki/Phagemid Does that help?

Main content

Overview: DNA cloning

Google Classroom

Definition, purpose, and basic steps of DNA cloning.

Key points:

DNA cloning is a molecular biology technique that makes many identical copies of a piece of DNA, such as a gene.
In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid.
The plasmid is introduced into bacteria via a process called transformation, and bacteria carrying the plasmid are selected using antibiotics.
Bacteria with the correct plasmid are used to make more plasmid DNA or, in some cases, induced to express the gene and make protein.

Introduction

When you hear the word “cloning,” you may think of the cloning of whole organisms, such as Dolly the sheep. However, all it means to clone something is to make a genetically exact copy of it. In a molecular biology lab, what’s most often cloned is a gene or other small piece of DNA.

If your friend the molecular biologist says that her “cloning” isn’t working, she's almost certainly talking about copying bits of DNA, not making the next Dolly!

Overview of DNA cloning

DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid. The insertion is done using enzymes that “cut and paste” DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of fragments from multiple sources.

Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the plasmid are selected and grown up. As they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains.

What is the point of making many copies of a DNA sequence in a plasmid? In some cases, we need lots of DNA copies to conduct experiments or build new plasmids. In other cases, the piece of DNA encodes a useful protein, and the bacteria are used as “factories” to make the protein. For instance, the human insulin gene is expressed in E. coli bacteria to make insulin used by diabetics.

As you may know if you have friends or family members who are diabetic, the hormone insulin plays an important role in human health. In a healthy person’s body, insulin is produced in the pancreas. It travels through the bloodstream and binds to cells of the body, allowing them to take up the sugar glucose.

In a person with type I diabetes, the cells of the pancreas that produce insulin are damaged or destroyed. People with type I diabetes must regularly inject themselves with insulin from another source to prevent dangerously high levels of blood sugar (and to allow sugar to enter the cells of the body as fuel).

For many years, all of the insulin used by type I diabetics came from the pancreases of pigs and cows that had been slaughtered for meat. The insulin molecule is similar between humans, pigs, and cows, so pig or cow insulin can substitute for human insulin in the bodies of type I diabetics.

Starting in the 1980s, however, scientists developed new techniques to make human insulin using Escherichia coli (E. coli) bacteria. Basically, they turned the bacteria into tiny insulin-producing factories. This recombinant insulin, or insulin made by combining DNA from multiple sources (humans and bacteria), is now the primary treatment used by type I diabetics.

Steps of DNA cloning

DNA cloning is used for many purposes. As an example, let's see how DNA cloning can be used to synthesize a protein (such as human insulin) in bacteria. The basic steps are:

Cut open the plasmid and "paste" in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA).
Insert the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up the plasmid.
Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein. Harvest the protein from the bacteria and purify it.

Let's take a closer look at each step.

1. Cutting and pasting DNA

How can pieces of DNA from different sources be joined together? A common method uses two types of enzymes: restriction enzymes and DNA ligase.

A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs. If two molecules have matching overhangs, they can base-pair and stick together. However, they won't combine to form an unbroken DNA molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone.

Our goal in cloning is to insert a target gene (e.g., for human insulin) into a plasmid. Using a carefully chosen restriction enzyme, we digest:

The plasmid, which has a single cut site
The target gene fragment, which has a cut site near each end

Then, we combine the fragments with DNA ligase, which links them to make a recombinant plasmid containing the gene.

2. Bacterial transformation and selection

Plasmids and other DNA can be introduced into bacteria, such as the harmless E. coli used in labs, in a process called transformation. During transformation, specially prepared bacterial cells are given a shock (such as high temperature) that encourages them to take up foreign DNA.

A plasmid typically contains an antibiotic resistance gene, which allows bacteria to survive in the presence of a specific antibiotic. Thus, bacteria that took up the plasmid can be selected on nutrient plates containing the antibiotic. Bacteria without a plasmid will die, while bacteria carrying a plasmid can live and reproduce. Each surviving bacterium will give rise to a small, dot-like group, or colony, of identical bacteria that all carry the same plasmid.

Not all colonies will necessarily contain the right plasmid. That’s because, during a ligation, DNA fragments don’t always get “pasted” in exactly the way we intend. Instead, we must collect DNA from several colonies and see whether each one contain the right plasmid. Methods like restriction enzyme digestion and PCR are commonly used to check the plasmids.

3. Protein production

Once we have found a bacterial colony with the right plasmid, we can grow a large culture of plasmid-bearing bacteria. Then, we give the bacteria a chemical signal that instructs them to make the target protein.

The bacteria serve as miniature “factories," churning out large amounts of protein. For instance, if our plasmid contained the human insulin gene, the bacteria would start transcribing the gene and translating the mRNA to produce many molecules of human insulin protein.

Humans and bacteria share the same genetic code, meaning that a human gene can be transcribed and translated in bacteria.

However, in order for a human gene to be expressed in bacteria, it must have one important modification. Specifically, it must lack introns. Introns are intervening sequences found in many human genes, and they are removed from eukaryotic RNA transcripts by splicing to make a mature mRNA. Bacteria do not have introns and cannot splice RNA transcripts.

How can we get a version of a human gene with the introns removed? An intron-less version of a human gene can be made from the mRNA that encodes the gene, using the enzyme reverse transcriptase. Reverse transcriptase synthesizes a DNA strand using an mRNA strand as a template. After an additional step to the DNA double-stranded, an intron-less version of the gene (called a cDNA) has been produced.

Once the protein has been produced, the bacterial cells can be split open to release it. There are many other proteins and macromolecules floating around in bacteria besides the target protein (e.g., insulin). Because of this, the target protein must be purified, or separated from the other contents of the cells by biochemical techniques. The purified protein can be used for experiments or, in the case of insulin, administered to patients.

Well...yes and no. The recombinant insulin made by pharmaceutical companies is in fact generated in bacteria, and it's expressed from a plasmid that bears a modified human insulin gene. The insulin made by the bacteria is purified using a form of column purification.

However, producing biologically active insulin requires some additional steps. Insulin is made as a single polypeptide chain. However, disulfide bonds must form within the chain, and part of it must be snipped out to form two separate chains (held together only by the disulfide bonds). Only then is the insulin biologically active, that is, able to act as a hormone in a patient's body.

Production of insulin that can be used by diabetics requires that these additional steps be incorporated into the protein purification procedure, so that the insulin protein takes on its correct three-dimensional form.

Uses of DNA cloning

DNA molecules built through cloning techniques are used for many purposes in molecular biology. A short list of examples includes:

Biopharmaceuticals. DNA cloning can be used to make human proteins with biomedical applications, such as the insulin mentioned above. Other examples of recombinant proteins include human growth hormone, which is given to patients who are unable to synthesize the hormone, and tissue plasminogen activator (tPA), which is used to treat strokes and prevent blood clots. Recombinant proteins like these are often made in bacteria.
Gene therapy. In some genetic disorders, patients lack the functional form of a particular gene. Gene therapy attempts to provide a normal copy of the gene to the cells of a patient’s body. For example, DNA cloning was used to build plasmids containing a normal version of the gene that's nonfunctional in cystic fibrosis. When the plasmids were delivered to the lungs of cystic fibrosis patients, lung function deteriorated less quickly $^{2}$ ‍.
Gene analysis. In basic research labs, biologists often use DNA cloning to build artificial, recombinant versions of genes that help them understand how normal genes in an organism function.
For instance, researchers studying neurons in fruit flies might use DNA cloning to assemble a reporter construct for a neural gene. In this construct, the regulatory region (promoter) of the gene might be pasted in front of a gene encoding a fluorescent protein. When transferred into a fly, the "reporter gene" would be expressed in the same neurons as the neural gene itself, causing those neurons to glow (fluoresce) under UV light.

These are just a few examples of how DNA cloning is used in biology today. DNA cloning is a very common technique that is used in a huge variety of molecular biology applications.

Explore outside of Khan Academy

Do you want to learn more about restriction enzymes? Check out this scrollable interactive and this simulation from LabXchange.

Do you want to learn more about the role of DNA ligase in gene cloning? Check out this scrollable interactive and this simulation from LabXchange.

Do you want to learn more about bacterial transformation? Check out this simulation from LabXchange.

Do you want to learn more about the selection of transformed bacteria? Check out this simulation from LabXchange.

LabXchange is a free online science education platform created at Harvard’s Faculty of Arts and Sciences and supported by the Amgen Foundation.

This article is licensed under a CC BY-NC-SA 4.0 license.

Works cited:

Superbest. (2014, June 11). How does heat shock transformation work? In Biology stack exchange. Retrieved from http://biology.stackexchange.com/questions/19038/how-does-heat-shock-transformation-work.
Alton, E. W. F. W., Armstrong, D. K., Ashby, D., Bayfield, K. J., Bilton, Diana, Bloomfield, E. V., ... Wolstenholme-Hogg, P. (2015). Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: A randomised, double-blind, placebo-controlled, phase 2b trial. Lancet Respiratory Medicine, 3(9), 684-691. http://dx.doi.org/10.1016/S2213-2600(15)00245-3.

Additional references:

Affibody. (n.d.). Insulin capture from human serum. Retrieved from http://affibody.com/upload/Research%20Reagents/Application%20notes/Insulin%20AFF%20application%20note%202007-03-30.pdf.

Amgen Biotech Experience. (2015). Student guide. Retrieved from https://www.amgenbiotechexperience.com/sites/abe.edc.org/files/abe_english_student_all_sequences_05.15.15.pdf.

Biotechnology. (2016, March 23). Retrieved March 29, 2016 from Wikipedia: https://en.wikipedia.org/wiki/Biotechnology.

Gebel, E. (2013). Making insulin: A behind-the-scenes look at producing a lifesaving medication. In Diabetes forecast. Retrieved from http://www.diabetesforecast.org/2013/jul/making-insulin.html?referrer=https://www.google.com/.

Gene therapy breakthrough for cystic fibrosis. (2015, July 3). In NHS choices. Retrieved from http://www.nhs.uk/news/2015/07July/Pages/Gene-therapy-breakthrough-for-cystic-fibrosis.aspx.

R&D Systems. (2016). Recombinant human growth hormone (GH) protein. In Growth hormone. Retrieved from https://www.rndsystems.com/products/recombinant-human-growth-hormone-gh-protein_1067-gh.

Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). DNA tools and biotechnology. In Campbell biology (10th ed., pp. 408-435). San Francisco, CA: Pearson.

Wilkin, D. (2016, March 23). Gene cloning - advanced. In CK-12 biology advanced concepts. Retrieved from http://www.ck12.org/book/CK-12-Biology-Advanced-Concepts/section/9.2/.

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Ilana Sacolick
Posted 5 years ago. Direct link to Ilana Sacolick's post “Why does the DNA need to ...”
Why does the DNA need to be in the form of a plasmid when pasted in a bacteria and not just the string of DNA cut by the restriction enzyme?
Button navigates to signup pageButton navigates to signup page
(13 votes)
Answer
- tyersome
  Posted 5 years ago. Direct link to tyersome's post “There are several reasons...”
  There are several reasons:
  
  1) Linear DNA is unstable because there are enzymes present within all organisms (including bacteria) that degrade linear DNA molecules.
  
  2) Vectors contain a sequence (known as the origin of replication) that causes the DNA to be replicated within the bacteria — this is necessary to maintain at least one copy of the new DNA per bacterium as the cells divide.
  
  3) Vectors usually contain at least one sequence that allows selection for the vector (e.g. antibiotic resistance) — this is discussed in this article.
  
  4) Vectors can also be used to do different things with the DNA. A common example of this would be an expression vector — this causes the DNA to be transcribed and translated and would allow you to examine the protein encoded in the cloned DNA.
  
  Does that help?
  Button navigates to signup page
  (31 votes)
Noor ul ain
Posted 8 years ago. Direct link to Noor ul ain's post “Are identical twins also ...”
Are identical twins also clones ?
Button navigates to signup pageComment on Noor ul ain's post “Are identical twins also ...”
(5 votes)
Answer
- Isabel Kaspriskie
  Posted 8 years ago. Direct link to Isabel Kaspriskie's post “The short answer is no. W...”
  The short answer is no. While they share a lot of DNA, there are also mutations that naturally occur within your cells, so no two people can share exactly the same DNA.
  Comment on Isabel Kaspriskie's post “The short answer is no. W...”
  (23 votes)
damiaR980
Posted 6 years ago. Direct link to damiaR980's post “When the bacteria taking ...”
When the bacteria taking up the plasmid. How can we be sure that the bacteria used aren’t having any plasmid in it? To make sure that when we grow it on agar the bacteria got the recombinant DNA. What if. The original bacteria we use to take up the recombinant plasmids are already having its own plasmid. So how can we differentiate them then?
Button navigates to signup pageButton navigates to signup page
(6 votes)
Answer
- tyersome
  Posted 6 years ago. Direct link to tyersome's post “Good question :-) It cou...”
  Good question :-)
  
  It could be difficult to know if you were just using a random bacteria isolated from nature — especially since there are likely to be many thousands of different plasmids (1730 were present in a sequence database as of 2009). We could sequence all the DNA inside the bacteria, but that is still a lot of work ...
  
  However that doesn't matter as much as you might think.
  
  For example, assume we are using a plasmid that contains a marker (selectable gene) encoding resistance to ampicillin. All we need to know is that the bacteria were are transforming are not already resistant to ampicillin. This is easy to test — we just try growing the bacteria in the presence of ampicillin, if they don't then we can use our plasmid.
  
  In practice microbiologists have domesticated strains of bacteria (a favorite is Escherichia coli — often abbreviated to E. coli) that have been studied for decades. In almost all cases you would be using one of these well characterized strains and so would not need to worry about whether there were unknown plasmids.
  Button navigates to signup page
  (13 votes)
Jousboxx
Posted 8 years ago. Direct link to Jousboxx's post “Do the bacteria ever make...”
Do the bacteria ever make mistakes in the replication process? If not, why? If they do, how can we still call Dolly the sheep a clone if the original sheep is actually slightly different?
Button navigates to signup pageComment on Jousboxx's post “Do the bacteria ever make...”
(5 votes)
Answer
- Chiara
  Posted 7 years ago. Direct link to Chiara's post “This is DNA cloning, not ...”
  This is DNA cloning, not the actual cloning of organisms. For more information on cloning, visit this webpage: https://en.m.wikipedia.org/wiki/Cloning. Hope you find it useful! :)
  Button navigates to signup page
  (5 votes)
Petra Yang
Posted 8 years ago. Direct link to Petra Yang's post “Why bacterias without pla...”
Why bacterias without plasmids will die?What are the functions of plasmid?
Button navigates to signup pageButton navigates to signup page
(3 votes)
Answer
- Chiara
  Posted 7 years ago. Direct link to Chiara's post “*Plasmids* are usually pr...”
  Plasmids are usually present in bacteria, and plasmids can replicate its own DNA independently of the bacteria, which is why it is often used in DNA cloning. Plasmids usually have an antibiotic resistant gene, so the bacteria won't die in the antibiotic. For more information, visit this webpage: https://en.m.wikipedia.org/wiki/Plasmid. I hope this answers your question! :)
  Comment on Chiara's post “*Plasmids* are usually pr...”
  (7 votes)
Isabella Sherring
Posted 6 years ago. Direct link to Isabella Sherring's post “How is the cutting and jo...”
How is the cutting and joining of DNA monitored?
Button navigates to signup pageButton navigates to signup page
(4 votes)
Answer
- tyersome
  Posted 6 years ago. Direct link to tyersome's post “To assess whether a "dige...”
  To assess whether a "digest" (restriction enzyme cutting reaction) is complete, we usually run a small sample of the digest on an agarose gel§ with a "ladder" sample containing fragments of known sizes and a small sample of uncut DNA.
  
  This comparison allows us to see whether we got fragments of the expected sizes and how much uncut plasmid still remains.
  
  We generally don't directly check whether a ligation has worked — ligation is very reliable and it is usually easier to just transform the DNA into a new host bacteria.
  We then purify plasmid DNA and use restriction digests, PCR †, or sequencing to test whether we got the desired outcome.
  
  §For details see:
  https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/v/gel-electrophoresis-dna
  
  †For details see:
  https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/v/the-polymerase-chain-reaction-pcr
  Button navigates to signup page
  (3 votes)
Muhammat Janabayevich
Posted 4 years ago. Direct link to Muhammat Janabayevich's post “How can we clone a gene t...”
How can we clone a gene that is unknown sequence ?
Button navigates to signup pageButton navigates to signup page
(4 votes)
Answer
- Ivana - Science trainee
  Posted 4 years ago. Direct link to Ivana - Science trainee's post “You can use partial diges...”
  You can use partial digestion method and expression in bacterial vectors and finally antibiotics to select your cloned vectors.
  
  Look at this:
  https://www.researchgate.net/post/How_do_i_clone_genes_with_unknown_sequence
  
  particularly this paper has protocol:
  https://www.researchgate.net/publication/10637344_Partial_characterization_of_a_transposon_containing_the_tetA_determinant_in_a_clinical_isolate_of_Acinetobacter_baumannii using tetA resistance gene as a marker on a plasmid (resistance to Tetracycline).
  Button navigates to signup page
  (2 votes)
Mishgan Fatima
Posted 5 years ago. Direct link to Mishgan Fatima's post “Could you have a vector o...”
Could you have a vector other than the bacterial plasmid
for instance a bacteriophage
Button navigates to signup pageButton navigates to signup page
(3 votes)
Answer
- tyersome
  Posted 5 years ago. Direct link to tyersome's post “Yes, though every case I ...”
  Yes, though every case I know of involves a phage based plasmid (known as a "phagemid") that is manipulated as a bacterial vector before being converted into a bacteriophage.
  
  I haven't personally used phagemids and suspect they are no longer commonly used, but you can learn more about this technology here:
  https://en.wikipedia.org/wiki/Phagemid
  
  Does that help?
  Button navigates to signup page
  (3 votes)
Zoe
Posted 3 years ago. Direct link to Zoe's post “I could find that DNA clo...”
I could find that DNA cloning and the virus reproduction are quite similar, in terms of that a cell accepts a new genetic material from outside, and the number of them increase. Is the virus reproduction of a human body associated with DNA cloning? How are they different?
Button navigates to signup pageButton navigates to signup page
(4 votes)
Answer
Zoe
Posted 3 years ago. Direct link to Zoe's post “There are two genetic mat...”
There are two genetic material, a plasmid and a bacterial chromosome in a bacterium. I think the bacterial chromosome also can produce protein for their metabolism, going through the transcription and translation. I'm just wondering about whether the bacterial chromosome in a bacterium works while another genetic material plasmid from outside works to produce proteins.
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(4 votes)
Answer

AP®︎/College Biology

Course: AP®︎/College Biology > Unit 6