Podcast
Questions and Answers
What is a notable characteristic of Bacillus subtilis that aids in its survival?
What is a notable characteristic of Bacillus subtilis that aids in its survival?
- It has a low gene count.
- It can form spores. (correct)
- It is incapable of producing secreted proteins.
- It has a double membrane.
Which promoter type is typically used in inducible expression systems?
Which promoter type is typically used in inducible expression systems?
- Lac promoter (correct)
- Constitutive promoter
- Constitutional promoter
- Basal promoter
What is a disadvantage of using bacterial expression systems?
What is a disadvantage of using bacterial expression systems?
- Limited gene expression
- Energy drain from strong expression (correct)
- Easy retention of plasmids
- Weak constitutional expression
E. coli differs from Bacillus subtilis by having how many membranes?
E. coli differs from Bacillus subtilis by having how many membranes?
What occurs during the growth phase of bacterial culture?
What occurs during the growth phase of bacterial culture?
What is indicated by the transformation of LR to BR in the context provided?
What is indicated by the transformation of LR to BR in the context provided?
Which components are involved in the conversion process mentioned in the content?
Which components are involved in the conversion process mentioned in the content?
What role do background growth conditions play in the process described?
What role do background growth conditions play in the process described?
What is the purpose of selecting AmpR colonies in this process?
What is the purpose of selecting AmpR colonies in this process?
What does the term 'toxin' refer to in the context of the provided information?
What does the term 'toxin' refer to in the context of the provided information?
What is a significant advantage of using Gateway cloning?
What is a significant advantage of using Gateway cloning?
What is required when performing a Gibson assembly?
What is required when performing a Gibson assembly?
Which of the following describes the role of restriction enzymes in the Gateway cloning process?
Which of the following describes the role of restriction enzymes in the Gateway cloning process?
How many fragments can be joined using the Gibson assembly technique?
How many fragments can be joined using the Gibson assembly technique?
What is the purpose of introducing att sequences into the vector during the cloning process?
What is the purpose of introducing att sequences into the vector during the cloning process?
Which of the following is NOT a characteristic of recombination cloning?
Which of the following is NOT a characteristic of recombination cloning?
Why is spectinomycin selection important in the cloning process described?
Why is spectinomycin selection important in the cloning process described?
What method is used to join DNA fragments in the described cloning techniques?
What method is used to join DNA fragments in the described cloning techniques?
What is the process of transduction in bacteria?
What is the process of transduction in bacteria?
Which of the following is NOT a step in the generation of a transgenic organism?
Which of the following is NOT a step in the generation of a transgenic organism?
What is one of the advantages of using E.coli as a host for protein expression?
What is one of the advantages of using E.coli as a host for protein expression?
Which of the following is a basic requirement for protein expression?
Which of the following is a basic requirement for protein expression?
What are the main components of a gene construct for protein expression?
What are the main components of a gene construct for protein expression?
Which type of cells is traditionally used to produce recombinant proteins?
Which type of cells is traditionally used to produce recombinant proteins?
What was the first licensed human drug produced using recombinant DNA technology in E.coli?
What was the first licensed human drug produced using recombinant DNA technology in E.coli?
Which expression system is NOT commonly used for producing recombinant proteins?
Which expression system is NOT commonly used for producing recombinant proteins?
What is a key advantage of using yeast as an expression system?
What is a key advantage of using yeast as an expression system?
What is a common disadvantage when expressing proteins in yeast?
What is a common disadvantage when expressing proteins in yeast?
In the baculovirus-insect cell expression system, what is the significance of the polyhedrin promoter?
In the baculovirus-insect cell expression system, what is the significance of the polyhedrin promoter?
Why is GAPD utilized in yeast expression systems?
Why is GAPD utilized in yeast expression systems?
How can yeast be stored for long-term use?
How can yeast be stored for long-term use?
What is a major challenge associated with the purification of proteins expressed in yeast?
What is a major challenge associated with the purification of proteins expressed in yeast?
Which statement reflects a misconception about recombinant products from yeast?
Which statement reflects a misconception about recombinant products from yeast?
What is a consideration when using baculovirus for protein expression?
What is a consideration when using baculovirus for protein expression?
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Study Notes
Gene Delivery in Bacteria
- Transduction is a natural process where viruses transfer genetic material between bacteria.
- Methods to introduce recombinant DNA into host cells include: isolating genes, fusing with vectors, delivering recombinant DNA, selecting modified hosts, and breeding for traits.
Protein Expression Systems
- Popular systems for recombinant protein production include bacteria, yeast, insect cells, and mammalian cells.
- Genetic engineering may alter biopathways in these expression systems.
Requirements for Protein Expression
- Essential components for protein expression include a gene construct (promoter, structural gene, terminator) and appropriate host cells like E. coli, Bacillus subtilis, yeast, insect cells, and mammalian cells.
Escherichia coli (E. coli)
- Fast-growing bacterium with a doubling time of 20 minutes.
- Contains around 4400 genes and can be preserved at -70°C.
- First licensed human drug from recombinant DNA, Humulin (insulin), was produced by E. coli in 1982.
Gene Expression Constructs
- Constructs for protein expression must contain elements that regulate transcription and terminate mRNA production.
- A strong promoter can lead to constitutive expression, which may strain the host.
Inducible Expression System
- The lac promoter system, when activated by IPTG, allows control over gene transcription at different growth phases.
Yeast as an Expression System
- Yeast like Saccharomyces cerevisiae is capable of stable plasmid maintenance and post-translational modifications.
- Uses essential genes for survival under selective conditions, like GAPD for leucine biosynthesis.
Advantages of Yeast Expression
- Established technology for large-scale fermentation with a proven safety record (GRAS status).
- Capable of post-translational modifications enhancing protein functionality.
Disadvantages of Yeast Expression
- Typically results in low levels of expressed proteins.
- Proteins may be retained in the periplasmic space, complicating purification processes.
Baculovirus-Insect Cell Expression System
- Utilizes baculovirus, specific to insects, to introduce genes into host cells.
- Uses strong Polyhedrin promoter to achieve high levels of mRNA expression, minimizing threats to humans.
Cloning Methods
- Techniques like Gibson assembly allow for the rapid joining of multiple DNA fragments for cloning purposes.
- PCR primers with overlapping sequences are essential for efficient assembly and cloning into vectors.
Bacillus subtilis
- Gram-positive bacterium with around 4100 genes, notable for spore-forming capabilities that enhance survival.
- Often utilized as an industrial host for secreted protein production due to its easier large-scale culturing processes.
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