Biotechnology Overview: Plasmids & Recombinant DNA

Plasmids

• Circular DNA molecule, separate from bacterial DNA
• Replicates independently
• Can be used to introduce foreign DNA into bacteria

Recombinant DNA Technology

• Used to study gene arrangement, expression and regulation
• Modifies gene expression to enhance or suppress products
• Creates artificial copies of DNA segments
• Transfers genes between organisms, creating transgenic organisms with altered characteristics
• Creates transgenic organisms used for commercial purposes

Restriction Endonucleases (REs)

• Bacterial enzymes that cut DNA at specific recognition sites
• Type II REs are used in gene cloning because they cleave DNA at specific sites
• Important for mapping and reconstructing DNA in vitro

Terminal Deoxynucleotidyl Transferase (Terminal Transferase)

• Enzyme that adds deoxynucleotides to the 3' end of DNA molecules
• Does not have proofreading ability
• Distinct from DNA polymerase III, which has proofreading ability

Foreign DNA Cloning Sources

• Chromosomal DNA
• mRNA converted to cDNA
• PCR-amplified DNA

Cloning and Expression Vectors

• Carry foreign DNA into bacterial cells
• Used in gene cloning and protein production
• Plasmids are named with a system of uppercase letters and numbers, "p" stands for plasmid
• pBR322, pUC18, p UC19 are examples of commonly used plasmids
• High copy number plasmids replicate more than 5oo copies per bacterial host cell, occur in plasmids with relaxed replication control
• Stringent replication is coupled to host chromosome, results in lower copy numbers

Essential features of a bacterial plasmid vector

• Origin of replication (ori) for independent replication
• Selection marker/s for identifying transformed cells:
  • Antibiotic resistance
  • Blue-white screening
• Multiple cloning site (MCS) containing restriction endonuclease cleavage sites for insertion of foreign DNA
• Easy extraction from host cells

Inserting Foreign DNA into Plasmid Vectors

• Single restriction approach:
  • Can lead to ligation of the restricted plasmid
  • Foreign DNA can be inserted in both orientations
  • Multiple fragments can insert into the excised plasmid
• Double restriction approach:
  • Uses two restriction enzymes to cleave the plasmid and foreign DNA components
  • Reduces problems associated with single restriction
  • Directional cloning, ensures foreign DNA is inserted in the correct orientation with respect to transcriptional and translational control sequences

Bacterial Transformation

• Process of introducing recombinant plasmids into bacterial cells
• Makes bacterial membrane transiently porous for plasmid uptake

Cosmids:

• Plasmids with a cos site (cohesive end site)
• Can be packaged into phage heads due to the cos site
• Can carry inserts of 33-47 kbp
• Combine advantages of plasmids and phage vectors

Bacterial Artificial Chromosomes (BACs):

• Based on the fertility plasmid (F plasmid)
• Designed for cloning large DNA sequences
• Contain partition genes for even distribution of plasmids during bacterial cell division
• Accommodate inserts of up to 300 kbp
• Used in chromosome mapping

Expression Vectors:

• Designed for protein expression of target genes
• Often plasmid-based
• Utilize T7 RNA polymerase gene expression system for efficient production of proteins
• Contain transcriptional and translational control sequences for gene expression
• Transform E.coli host cells with a T7 RNA polymerase gene integrated into its chromosome

Explore the fundamental concepts of plasmids, recombinant DNA technology, and the role of restriction endonucleases. This quiz covers how these elements work together to modify gene expression and create transgenic organisms for various applications. Test your understanding of key enzymes and their functions in molecular biology.