Summary of Recombinant DNA Technology - Animated Video

Summary of "Recombinant DNA Technology - Animated Video"

This video provides a detailed overview of Recombinant DNA Technology, explaining the step-by-step methodology used to clone and express a gene of interest, such as the Insulin gene, in bacterial cells. It covers the entire process from gene selection to protein purification, highlighting key enzymes, vectors, and techniques involved.

---

Main Ideas and Concepts

• Applications of Recombinant DNA Technology:
  Widely used in medicine, agriculture, and biotechnology for producing desired proteins.
• Gene of Interest Preparation:
  
  • Selection of a gene coding for the desired protein (e.g., Insulin).
  • Gene can be chemically synthesized or obtained from complementary DNA (cDNA) libraries.
  • cDNA libraries are constructed by isolating mRNA, using oligo(dT) primers to bind poly-A tails, and synthesizing cDNA via reverse transcriptase and DNA polymerase.
  • Sequencing confirms the gene’s identity before cloning.
• Preparation of Transfer Vector:
  
  • Vectors (Plasmids, cosmids, viral vectors, artificial chromosomes) carry the gene into host cells.
  • Plasmids are the most common vectors, containing an origin of replication, antibiotic resistance gene (e.g., ampicillin resistance), and multiple cloning sites (polylinker) within the lacZ gene.
• Restriction Enzyme Digestion:
  
  • Restriction Enzymes cut DNA at specific sequences, producing sticky or blunt ends.
  • Both the gene of interest and plasmid vector are cut with the same Restriction Enzymes to create compatible ends for ligation.
• Ligation of DNA Molecules:
  
  • DNA Ligase enzyme joins the gene insert and plasmid vector by forming phosphodiester bonds, requiring ATP.
• Transformation of Host Cells:
  
  • Chemical transformation method using calcium chloride and competent E. coli cells.
  • Calcium ions neutralize negative charges on DNA and bacterial membranes, facilitating DNA binding.
  • Heat shock at 42°C creates temporary pores in bacterial membranes allowing plasmid entry.
  • Recovery phase in nutrient-rich medium allows cells to repair and express antibiotic resistance.
• Bacterial Cultivation and Selection:
  
  • Transformed bacteria are grown on antibiotic-containing media (e.g., ampicillin) to select for cells carrying the plasmid.
  • Three outcomes: no plasmid uptake (cells die), uptake of unaltered vector, or uptake of recombinant vector.
  • Blue-white screening using X-gal substrate distinguishes colonies:
    • Blue colonies = bacteria with unaltered vector (functional lacZ gene).
    • White colonies = bacteria with recombinant vector (lacZ disrupted by gene insert).
• Isolation and Growth of Recombinant Bacteria:
  
  • White colonies are isolated and cultured in enriched media.
  • Small-scale growth for lab use or large-scale growth in bioreactors for industrial protein production.
• Protein Purification:
  
  • If protein is intracellular, bacterial cells are harvested by centrifugation and lysed (e.g., enzymatic lysis buffer).
  • Chromatography (such as affinity chromatography) purifies the target protein by binding it to a stationary phase and washing away impurities.
  • Elution releases the purified protein, making it ready for use.

---

Detailed Methodology (Step-by-Step)

1. Gene Preparation:
   
   • Isolate mRNA from cells.
   • Use oligo(dT) primers to bind poly-A tail.
   • Reverse transcribe mRNA to single-stranded cDNA.
   • Synthesize second DNA strand to form double-stranded cDNA.
   • Sequence cDNA and construct cDNA library.
2. Vector Preparation:
   
   • Choose appropriate vector (commonly Plasmids like pUC8).
   • Identify and prepare multiple cloning sites within lacZ gene.
3. Restriction Enzyme Digestion:
   
   • Treat gene insert and plasmid with the same Restriction Enzymes to create compatible ends.
4. Ligation:
   
   • Mix cut gene and plasmid with DNA Ligase and ATP.
   • Ligase catalyzes phosphodiester bond formation, joining insert and vector.
5. Transformation:
   
   • Prepare competent E. coli cells using calcium chloride.
   • Mix recombinant plasmid with competent cells on ice.
   • Heat shock at 42°C for 90 seconds to allow DNA uptake.
   • Return to ice, then add recovery medium and incubate at 37°C for 1 hour.
6. Selection and Screening:
   
   • Plate bacteria on ampicillin-containing medium with X-gal.
   • Incubate overnight at 37°C.
   • Select white colonies (recombinant plasmid present).
7. Cultivation:
   
   • Inoculate selected colonies into enriched medium for protein production.

Category

Educational

Video