Subtopic 1: Restriction Endonucleases

Restriction enzymes are sequence-specific endonucleases found in bacteria that cleave dsDNA at defined recognition sequences. They act as a bacterial immune mechanism against bacteriophage DNA. Recognition sequences are typically palindromic (4–8 bp). EcoRI recognizes 5'-GAATTC-3' and makes a staggered cut between G↓A on both strands, leaving 4-nucleotide 5' overhangs (sticky ends). Enzymes that cut both strands at the same position produce blunt ends. More than 900 enzymes have been identified from over 230 bacterial strains. DNA ligase seals phosphodiester bonds to join fragments.

Subtopic 2: Cloning Vectors

A cloning vector must have: ori (autonomous replication), selectable markers (antibiotic resistance), and cloning/restriction sites. pBR322 possesses ampR and tetR markers; insertional inactivation of tetR identifies recombinants. The Ti plasmid of Agrobacterium tumefaciens is the principal plant transformation vector — its T-DNA naturally integrates into the plant genome; disarming removes oncogenic sequences. Lambda phage serves as a vector for larger inserts (up to ~23 kb) in E. coli.

Subtopic 3: Recombinant DNA Construction and Host Introduction

Recombinant DNA is created by digesting vector and insert with the same enzyme, then ligating with DNA ligase. Host cells must be made competent: $CaCl_{2}$ treatment followed by heat shock (42 °C, 60–90 s) for bacteria. Physical methods include microinjection (nucleus delivery), biolistics (gene gun with gold/tungsten particles), and electroporation (electric-pulse-mediated pore formation). Disarmed Ti plasmid and retroviral vectors extend the host range.

Subtopic 4: PCR

PCR requires template DNA, sequence-specific forward and reverse primers, four dNTPs, thermostable Taq polymerase, and $MgCl_{2}$. The three-step cycle — denaturation (94–98 °C), annealing (50–65 °C), extension (72 °C) — is repeated 25–40 times for exponential amplification (2ⁿ copies). Taq polymerase, derived from Thermus aquaticus (hot springs), withstands denaturation temperatures.

Subtopic 5: Gel Electrophoresis

DNA (negatively charged) is driven through agarose gel toward the anode under electric current. Smaller fragments migrate faster (farther from well). Ethidium bromide staining reveals orange bands under UV. Bands are compared to a standard DNA ladder to determine fragment sizes.

Subtopic 6: Bioreactors

Bioreactors maintain optimal conditions (temperature, pH, $O_{2}$, mixing) for large-scale cell culture. The stirred-tank design features an agitator and sparger. After biosynthesis, downstream processing (separation, purification, formulation, quality testing) yields the final product (e.g., recombinant insulin, vaccines).