Recombinant Plasmids

Recombinant Plasmids

Focus on Human Insulin Production

VCAA Key Knowledge

  • The use of recombinant plasmids as vectors to transform bacterial cells demonstrated by the production of human insulin.

Recombinant DNA Technology

  • Definition: Recombinant DNA technology refers to methods that involve the ‘recombining’ of DNA from different individuals and even different species.

  • Often referred to as recombinant DNA technology.

  • Use of Restriction Enzymes: Restriction enzymes can cut DNA (from different sources) into pieces that are easier to recombine in a test tube.

  • DNA Ligase Function: DNA ligase is used to repair gaps in the sugar-phosphate backbone of the DNA molecules.

  • Vectors Defined: Methods have been developed to insert recombinant DNA into cells using vectors, which are self-replicating DNA molecules that serve as carriers to transmit genes from one organism to another.

What are Plasmids?

  • Definition of Plasmids: Plasmids are small circular DNA molecules that act as vectors, carrying genes of interest into bacteria.

  • Commonly Used Cloning Vectors: The most commonly used cloning vectors are E. coli plasmids.

  • Functional Regions of Plasmids: All plasmids include three functional regions:

    1. An origin of replication.

    2. A drug-resistance gene.

    3. A region where DNA can be inserted without interfering with plasmid replication or expression of the drug-resistance gene.

  • Recombinant Plasmids: A recombinant plasmid is a plasmid that has had a DNA sequence (gene of interest) inserted into it.

Making a Recombinant Plasmid

Transforming Bacteria

  • Basic Process:

    • Components Involved:

    • Gene of Interest

    • Recombinant Plasmid

    • Transformed Bacteria

    • Plasmid Vector (circular DNA)

    • Untransformed Bacteria

    • Process Stages: The transformed bacteria go on to be cultured, where they transcribe and translate the gene of interest, producing proteins that scientists can harvest.

Proteins Made from Transformed Bacteria

  • Examples of proteins that can be produced from transformed bacteria:

    • Insulin: Manages diabetes.

    • Erythropoietin: For treatment of anaemia.

    • Growth Hormone: Manages growth disorders.

    • Interferon: For treatment of some cancers.

    • Hepatitis B Surface Antigen: Used in hepatitis B vaccine.

    • Chymosin: Used for cheese production.

    • Alpha-Amylase: Used for ethanol and high fructose corn syrup production.

Steps in Bacterial Transformation

  1. Prepare Transformation Solution: Treat bacteria with transformation solution (calcium chloride).

    • The positive charge of Ca++ ions binds the negatively charged cell membrane, giving it a positive charge.

    • When recombinant DNA plasmids are added, they are attracted to the cell membrane.

  2. Incubate on Ice: This decreases the fluidity of the cell membrane.

  3. Heat-Shock at 42°C: This increases the permeability of membranes so plasmids can enter the cells.

  4. Nutrient Broth Incubation at 37°C: Provides nutrients that allow bacteria to grow and express proteins before they are streaked onto selective media.

Selection and Screening of Bacterial Colonies

  • Selective Media Plating: Plating the transformed bacteria on selective media containing antibiotics ensures that only those bacteria carrying the plasmid with the appropriate antibiotic resistance gene will grow.

  • Identifying Transformed Bacteria: Transformed bacteria are identified by their reporter gene:

    • Beta-Galactosidase: In the case of the LacZ plasmid.

    • Green Fluorescent Protein: In the case of the pGLO plasmid.

  • Confirmation Methods: Restriction digests, PCR, or DNA sequencing can confirm that the plasmid is recombinant.

  • Potential Issues: It is possible for the cut plasmid to ligate back to itself rather than taking up the gene of interest, or the gene may be inserted in the wrong direction.

Vector Preparation

  • Restriction Digestion:

  • Dephosphorylation:

  • Blunt-End Creation (Optional):

  • Purification:

  • Insert Preparation Steps:

    • Restriction digestion.

    • Blunt-end creation (optional).

    • Purification.

  • Ligation Process:

    • Involves T4 DNA Ligase.

    • Includes factors like PEG, vector to insert ratio, reaction time, and temperature, with optional purification/heat-inactivation.

  • Transformation Efficiency Factors:

    • Choice of competent cells (chemical and electrocompetent cells).

  • Colony Screening:

    • Use blue/white colonies for positive selection, along with restriction digestion and colony PCR.

    • Sanger sequencing.

LacZ Plasmid

  • Features:

    • Beta Lactamase (bla) Gene: Codes for a protein that inactivates the antibiotic ampicillin.

    • lacZ Gene: Contains coding information for the first 146 amino acids of β-galactosidase.

    • Color Indicator: If not disrupted by the insertion of foreign DNA, bacterial colonies will express β-galactosidase and will turn blue when grown in the presence of the substrate X-gal.

pGLO Plasmid

  • Features:

    • Beta Lactamase (bla) Gene: Codes for a protein that inactivates antibiotic ampicillin.

    • Green Fluorescent Protein (GFP) Gene: Gene from Aequorea victoria jellyfish; fluoresces green after absorbing UV or blue light.

    • Regulatory Gene (araC): Protein that regulates GFP transcription (turns on GFP).

Screening Colonies Transformed with LacZ Plasmid

  • Process:

    • Combine plasmid vector with lacZAM15 and competent E. coli cultured with X-Gal + IPTG.

    • Insertion within lacZ leads to white colonies, while foreign DNA inserted outside lacZ leads to blue colonies.

Screening Colonies Transformed with pGLO Plasmid

  • Plating Conditions Example:

    • LB and antibiotic media with/without pGLO (results measuring colony counts).

Screening Colonies Using PCR

  • Molecular Weight Analysis:

    • Positive colonies have a molecular weight of 2072 bp.

    • Negative colonies have a molecular weight of 643 bp. Abbreviations correspond to:

    • M: Molecular weight marker

    • N: Negative control (PCR amplification of a colony from the transformation of the vector alone)

    • B: PCR blank control.

Structure of Human Insulin

  • Composition: Human insulin consists of two polypeptide chains known as the alpha and beta subunits.

  • Gene Coding: Each chain is coded by a different gene.

  • Functional Formation: The two chains join together by two disulfide bonds to form a functional insulin molecule.

Production of Human Insulin

  • Gene Amplification: The genes coding for the alpha and beta subunits of insulin contain introns, which must be removed because bacteria cannot splice these out. The gene for each subunit is thus amplified by RT-PCR.

  • Restriction Enzyme Cutting: Each PCR product is cut with distinct restriction enzymes that generate sticky ends, and the plasmids are also cut with these enzymes to ensure alignment with cut plasmids.

  • Mixing Conditions: After incubating together with DNA ligase, the recombinant plasmids are used to transform bacteria.

Production of Human Insulin Continued

  • Cultivation of Transformants: Successfully transformed bacteria are placed in conditions for exponential reproduction.

  • Protein Harvesting: As they grow, the bacteria produce human insulin proteins. Once cells reach optimal density, they are filtered from the growth medium and lysed to release the insulin proteins.

  • Purification: The A and B subunit proteins are purified from their respective bacterial sources and mixed together to allow disulfide bond formation, producing functional human insulin.

Key Steps in Insulin Production

  1. Insert genes for the two insulin polypeptides next to a highly expressed gene for β-galactosidase to form fusion proteins.

  2. Transform E. coli with the recombinant expression vectors, selecting transgenic cells by antibiotic resistance.

  3. Purify the fusion proteins.

  4. Chemically remove insulin polypeptides from the β-galactosidase protein (linking disulfide bridges).

  5. Combine the polypeptides to produce functional insulin.