Lecture 4 Part 2 Flashcards
What is recombinant DNA technology?
Artificially combining genetic material from different sources to form new DNA sequences not naturally occurring in that combination.
List three applications of recombinant DNA technology.
DNA Cloning, Protein Production, Genetic Modifications.
What is DNA cloning?
The process of isolating a DNA fragment from any species and propagating it without altering the original sequence.
What are the essential purposes of DNA cloning?
Generating multiple copies of DNA for genetic analysis.
Expressing the resulting protein for research or commercial use.
Manipulating DNA sequences to alter gene expression and protein function.
What is the first step in the DNA cloning process?
Isolation of DNA Fragments.
What is a vector in DNA cloning?
A DNA molecule that facilitates the transfer of genetic material into a host cell.
What are the key features of bacterial plasmid vectors?
Enable autonomous replication within bacterial cells.
Contain selectable markers such as antibiotic resistance genes.
Possess multiple cloning sites (MCS) for restriction enzymes.
May include regulatory sequences like promoters for controlled gene expression.
What is the role of restriction enzymes in DNA cloning?
They cut DNA at specific sequences, creating either blunt or sticky ends.
What is ligation in the context of DNA cloning?
The process where the enzyme DNA ligase catalyzes the covalent linkage between the 3′ and 5′ ends of DNA strands.
What are the two primary methods for transforming host cells with recombinant DNA?
Heat Shock, Electroporation.
Fill in the blank: The recombinant plasmid is introduced into host bacterial cells via _.
Transformation.
What does the term ‘transformation’ refer to in DNA cloning?
The introduction of recombinant plasmid into host bacterial cells.
What is the function of selectable markers in vectors?
To allow for screening of host cells containing the desired recombinant plasmid.
Molecular mechanisms in DNA cloning include…
restriction digestion and transformation.
What happens to bacterial cells after transformation?
Only a subset of bacterial cells will successfully take up the recombinant plasmid.
What is the purpose of antibiotic resistance selection in recombinant cloning?
To allow only transformed bacteria to survive when grown on antibiotic-containing media.
What role does X-gal play in blue-white screening?
It is a chromogenic substrate included in the growth medium.
What indicates a successful insertion of foreign DNA in blue-white screening?
White colonies indicate successful insertion, while blue colonies do not.
What technique is used to confirm the presence of recombinant DNA directly from bacterial colonies?
Colony PCR.
What is the purpose of restriction digestion analysis?
To verify the expected fragment sizes of isolated plasmid DNA.
What is used to ensure the correct insert is present and in the proper orientation?
DNA Sequencing.
What is necessary for producing the corresponding protein after inserting a recombinant DNA sequence into a vector?
Controlled gene expression.
What are expression vectors?
Vectors that include additional regulatory elements for gene expression.
List three regulatory elements included in expression vectors.
Promoters to initiate transcription.
Ribosome Binding Sites (RBS) for translation initiation.
Affinity Tags (e.g., His-tag) for simplified protein purification.
What is the purpose of polyhistidine tags in recombinant proteins?
For selective purification of proteins.
What method is used to purify proteins based on their affinity for metal ions?
Immobilized Metal Affinity Chromatography (IMAC).
What is the role of microbiology in recombinant DNA technology?
It is critical in the use of bacterial systems for genetic manipulation.
What are plasmids?
Circular DNA molecules naturally present in bacterial cells.
What do engineered plasmids serve as in molecular biology?
Essential tools for cloning, gene expression, and protein production.
What are competent cells?
Bacterial cells treated to become more permeable to DNA uptake.
What are two methods used to prepare competent cells?
Chemical treatments (calcium chloride), Physical methods (electroporation).
Name one application of recombinant bacteria in biotechnology.
Production of insulin, growth hormones, and industrial enzymes.
What is one environmental application of recombinant bacteria?
Engineering bacteria for bioremediation.
What is a significant agricultural application of recombinant bacteria?
Development of genetically modified crops for increased yield and pest resistance.
