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Wize University Biology Textbook > Lab Techniques for Biology

Plasmids & Restriction Enzymes

Gene EditingPrevious Section
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Overview of Gene Cloning

Gene cloning can be done when scientists want a gene to be expressed in a bacterial host. This is done in a series of steps:
  1. Amplify gene of interest
  2. Put gene in plasmid
  3. Put plasmid in bacteria
  4. Bacteria expresses gene
  5. Purify protein


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Step 1: PCR with restriction sites

Restriction sites are sequences of DNA that are recognized by specific enzymes (endonucleases). These sites can be added to the gene of interest by designing primers.
  • Primers partly match the DNA to be amplified.
  • The "hanging end" has the sequence we want to introduce and ends up amplified during PCR.

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Step 2: Digestion and ligation

Plasmid and PCR product are cut with restriction enzymes or endonucleases (REs).
  • Digestion with restriction enzymes creates complementary sticky ends.
  • DNA ligase sticks them together.

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Steps 3 & 4: Introduction of DNA in bacteria and gene expression.

Plasmids are inserted in bacterial cells.
  • Bacteria transcribe and translate the gene.
  • Protein is produced and can be purified.

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Plasmids

Plasmids are circular pieces of DNA that can replicate in bacteria. They are very often used as tools in biology and can be expressed by eukaryotic cells as well depending on the promoter present.
  • A gene of interest can be ligated into a plasmid using restriction sites.
  • When these are designed for bacteria, there is often an antibiotic resistance gene present that confers the bacteria which have the plasmid resistance to a particular antibiotic.
  • An Ori is also needed for replication.



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Restriction Enzymes or Endonucleases (REs)

Recognize and cut DNA at specific restriction sites which are often palindromic.
  • Originally from bacteria, they were enzymes used by bacteria to cut viral DNA.
  • Each restriction enzyme cuts at a specific DNA sequence.
  • They can create "blunt" or "sticky" ends. Example:

5'.....C TCGAG.....3' 5'.....GCC GGC.....3'

3'.....GAGCT C.....5' 3'.....CGG CCG.....5'

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  • To insert a gene into a plasmid, you need to introduce RE sites to the ends of the gene (by PCR).
  • These sites must match RE sits in the plasmid of interest.
  • Complementary sequences will be fused by DNA ligase.


Extra Practice
What is the main disadvantage of using only one blunt end restriction enzyme in cloning?
Suppose you want to clone a certain gene of interest into a vector as shown below. Which restriction enzyme(s) should you use? Which restriction enzyme sites will be removed from the vector's polylinker?
Plasmid
You are attempting to construct a restriction map for a plasmid. You digest the plasmid with the restriction enzymes EcoR1 and Bbs1 and run the fragments through gel electrophoresis. Given the following gel results, which if the correct restriction map?
You are attempting to construct a restriction map for a plasmid. You digest the plasmid with the restriction enzymes EcoR1 and Bbs1 and run the fragments through gel electrophoresis. Given the following gel results, which if the correct restriction map?
In Homolgous Recombination in diploid yeast cells, why is it important to replace one copy of the gene you are studying with a gene for a selectable marker?
Plasmid
Cloning Plasmids
What is NOT a common feature found in in every plasmid?
You are attempting to construct a restriction map for a plasmid. You digest the plasmid with the restriction enzymes EcoR1 and Bbs1 and run the fragments through gel electrophoresis. Given the following gel results, which if the correct restriction map?
Imagine you want to insert a foreign gene into a plasmid to over express this gene. In order to do this successfully, what must both the plasmid and your gene have?
Imagine you are a microbiologist growing different strains of bacteria in your lab. You
have the following flasks:
i. Flask 1 contains strain A and no antibiotics
You are studying an olfactory receptor in two strains the roundworm C. elegans. In the wildtype strain, the worms are able to smell attractive food odors. In the mutant strain, the worms are unable to sense when food is nearby. You believe that a severe DNA mutation has led to a deletion of the olfactory receptor gene from the mutant C. elegans worm's genome. How would you detect whether this specific DNA sequence is present in the mutant C. elegans genome?
Cloning Question
Suppose you want to clone a certain gene of interest into a vector as shown below. Which restriction enzyme(s) should you use? Which restriction enzyme sites will be removed from the vector's polylinker?
Which of the following statements is FALSE regarding genomic DNA libraries?
What is the main disadvantage of using only one blunt end restriction enzyme in cloning?
Suppose you want to clone a certain gene of interest into a vector as shown below. Which restriction enzyme(s) should you use? Which restriction enzyme sites will be removed from the vector's polylinker?