BIO 2.6 - Recombinant Plasmids

Steps of gene transfer in prokaryotes

1. DNA from bacterial cells, called plasmids (a ring of DNA) are cut at a specific site by a restriction endonuclease, creating sticky ends

2. A small length of DNA containing the target gene is cut using the same restriction endonuclease to create complementary sticky ends

3. The DNA is incorporated into the plasmid by matching the sticky ends together

4. DNA ligase catalyses the formation of phosphodiester bonds to join the DNA with the plasmid, creating recombinant DNA

5. The recombinant DNA is added back into the bacteria through transformation (heat shock or electroporation)

Steps of gene transfer in eukaryotes

1. Eukaryotes have introns in their DNA, so mRNA must be used to start the process

2. Reverse transcriptase binds to the mRNA and forms a complementary DNA strand (cDNA)

3. DNA polymerase binds to the template DNA strand to construct the second strand to form double stranded cDNA

4. The cDNA is cut with the same restriction endonuclease as the plasmid to create complementary sticky ends

5. The DNA is incorporated into the plasmid by matching the sticky ends together

6. DNA ligase catalyses the formation of phosphodiester bonds to join the DNA with the plasmid, creating recombinant DNA

7. The recombinant DNA is added back into the bacteria through transformation

Heat shock

1. Bacteria, recombinant and non-recombinant plasmids are placed with calcium ions in an ice cold solution

2. The solution is heated rapidly, causing the plasma membrane to disrupt, allowing the plasmids to enter the bacteria cells

Electroporation

Bacteria, recombinant and non-recombinant plasmids are subjected to an electric current, disrupting the plasma membrane, allowing the plasmids to enter the bacterial cells.

How are transformed bacteria identified?

• The recombinant plasmids contain an antibiotic resistant gene

• Bacteria are incubated so they can multiply, then they are plated on antibiotic agar plate

• Transformed bacteria survive on the agar plate due to the antibiotic resistant gene while bacteria that are not transformed will die

Steps of gene transfer for human insulin production (6)

1. The mature mRNA strand for Chain A is isolated from beta cells from the pancreas

2. Reverse transcriptase is added and builds a DNA strand complementary to the mRNA strand

3. DNA polymerase is added to build a second DNA strand, completing the double stranded cDNA

4. The cDNA is cut with a restriction endonuclease (EcoR1), leaving sticky ends

5. Plasmids containing the antibiotic resistance gene AMP^R (ampicillin resistant) and the LacZ gene which codes for the enzyme β-galactosidase are isolated from E.coli and cut with the same endonuclease (EcoR1), creating matching sticky ends.

6. The matching sticky ends of the Chain A gene and plasmid are joined together with DNA ligase to form phosphodiester bonds, producing a recombinant plasmid.

Steps of transformation of bacteria for human insulin production

• Heat shock or electroporation are used to transfer the recombinant plasmids into bacteria cells

• A mixture is formed, consisting of:

  • Bacteria that have not been transformed

  • Bacteria with non-recombinant plasmids

  • Bacteria with recombinant plasmids with the Chain A gene

Identifying transformed bacteria for human insulin production

Plate the bacteria on ampicillin and X-gal. X-gal is the substrate for the enzyme β-galactosidase. The enzyme breaks down X-gal into a product that turns the colonies blue.

• Bacteria not transformed die

• Bacteria that have been transformed but do not have the Chain A gene turn blue because a functional lacZ gene is present, allowing β-galactosidase to break down X-gal and produce blue colonies

• Bacteria with the Chain A gene produce white colonies because the Chain A gene is inserted into the lacZ gene, making a non-functioning β-galactosidase enzyme

Steps to produce human insulin after transformation

1. Transformed bacteria are incubated to produce lots of the desired product (Chain A joined to β-galactosidase)

2. The insulin Chain A polypeptides are separated from β-galactosidase

3. The whole process is repeated to produce Chain B polypeptides

4. The two polypeptide chains are joined together by disulfide bonds to form the functional insulin protein