Podcast
Questions and Answers
The ______ gene provides resistance to the antibiotic ampicillin.
The ______ gene provides resistance to the antibiotic ampicillin.
Ampicillin resistance
The ______ terminator signals the end of transcription for T7 RNA polymerase.
The ______ terminator signals the end of transcription for T7 RNA polymerase.
T7
The ______ ori is the origin of replication from the pBR322 plasmid.
The ______ ori is the origin of replication from the pBR322 plasmid.
pBR322
The Rop gene codes for proteins involved in the ______ of the plasmid.
The Rop gene codes for proteins involved in the ______ of the plasmid.
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The LacI protein represses transcription of the gene of interest from the ______ promoter.
The LacI protein represses transcription of the gene of interest from the ______ promoter.
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E. coli is particularly popular when the aim of the cloning experiment is to study the basic features of ______ biology.
E. coli is particularly popular when the aim of the cloning experiment is to study the basic features of ______ biology.
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Cloning vectors for ______ are used when the aim of the experiment is to study gene structure and function.
Cloning vectors for ______ are used when the aim of the experiment is to study gene structure and function.
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The pET expression vector is approximately ______ KB in size.
The pET expression vector is approximately ______ KB in size.
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In Gateway cloning, a ______ is moved into a vector through a two-step recombination process.
In Gateway cloning, a ______ is moved into a vector through a two-step recombination process.
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Lambda phage utilizes recombination machinery to integrate its genome into the host bacterial ______ during the lysogenic cycle.
Lambda phage utilizes recombination machinery to integrate its genome into the host bacterial ______ during the lysogenic cycle.
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The integration reactions in Gateway cloning are mediated by a site-specific ______ system derived from bacteriophage lambda.
The integration reactions in Gateway cloning are mediated by a site-specific ______ system derived from bacteriophage lambda.
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The recombination of attP and attB sites creates ______ and attR sites.
The recombination of attP and attB sites creates ______ and attR sites.
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The Gateway system involves two key ______ sites: attB and attP.
The Gateway system involves two key ______ sites: attB and attP.
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The BP reaction is involved in the creation of ______ clones.
The BP reaction is involved in the creation of ______ clones.
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The LR reaction takes place between the attL sites of the generated ______ clone and the attR sites of the destination vector.
The LR reaction takes place between the attL sites of the generated ______ clone and the attR sites of the destination vector.
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Gateway vectors contain modified versions of the att sites so that scientists can easily clone in their desired ______ sequences.
Gateway vectors contain modified versions of the att sites so that scientists can easily clone in their desired ______ sequences.
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Hydrogen bonding and ______ interactions occur between proteins and DNA.
Hydrogen bonding and ______ interactions occur between proteins and DNA.
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Open chromatin regions have ______ accessibility to transcription initiation complex.
Open chromatin regions have ______ accessibility to transcription initiation complex.
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The stable promotor sequence is characterized by a ______ rich sequence.
The stable promotor sequence is characterized by a ______ rich sequence.
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Transcription factors bind to specific ______ in the DNA known as a consensus site.
Transcription factors bind to specific ______ in the DNA known as a consensus site.
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The rate of peptide chain elongation increases with respect to an increase in ______.
The rate of peptide chain elongation increases with respect to an increase in ______.
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Strong promoters have high affinity for ______ polymerase, leading to efficient recruitment and initiation of transcription.
Strong promoters have high affinity for ______ polymerase, leading to efficient recruitment and initiation of transcription.
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Epigenetic modifications include ______ methylation and histone acetylation.
Epigenetic modifications include ______ methylation and histone acetylation.
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At higher ______, the rate of cHSPA6 synthesis was faster due to an increase in plasmid copy number, transcription rate, and peptide chain elongation rate.
At higher ______, the rate of cHSPA6 synthesis was faster due to an increase in plasmid copy number, transcription rate, and peptide chain elongation rate.
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Every protein folds differently depending upon its characteristics such as ______, hydrophobicity, post-translational modifications, isoelectric points, etc.
Every protein folds differently depending upon its characteristics such as ______, hydrophobicity, post-translational modifications, isoelectric points, etc.
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Thermus thermophilus, an extremophile, has a ______ rich genome to tolerate high environmental temperatures.
Thermus thermophilus, an extremophile, has a ______ rich genome to tolerate high environmental temperatures.
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The presence and affinity of transcription factor ______ sites influence the recruitment of RNA polymerase and the efficiency of transcription initiation.
The presence and affinity of transcription factor ______ sites influence the recruitment of RNA polymerase and the efficiency of transcription initiation.
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The final growth varied due to different ______ rates under each media.
The final growth varied due to different ______ rates under each media.
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TB medium showed the highest ______ rate under uninduced conditions.
TB medium showed the highest ______ rate under uninduced conditions.
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The induced culture in the TB medium expressing cHSPA6 showed the lowest ______ yield.
The induced culture in the TB medium expressing cHSPA6 showed the lowest ______ yield.
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The LB media showed relatively higher ______ rate under induced culture conditions resulting in higher volumetric yield.
The LB media showed relatively higher ______ rate under induced culture conditions resulting in higher volumetric yield.
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If the rate of protein synthesis supersedes ______ machinery, the recombinant protein will not fold correctly.
If the rate of protein synthesis supersedes ______ machinery, the recombinant protein will not fold correctly.
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Promoters may contain additional regulatory elements such as ______ and silencers, which modulate gene expression in response to cellular signals or environmental cues.
Promoters may contain additional regulatory elements such as ______ and silencers, which modulate gene expression in response to cellular signals or environmental cues.
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The presence and activity of these elements can influence ______ strength by affecting the rate of transcription initiation.
The presence and activity of these elements can influence ______ strength by affecting the rate of transcription initiation.
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Consult published literature and databases to identify ______ with proven performance in specific host systems.
Consult published literature and databases to identify ______ with proven performance in specific host systems.
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Experiment with different ______ to determine their suitability for driving protein expression in your specific application.
Experiment with different ______ to determine their suitability for driving protein expression in your specific application.
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Use bioinformatics tools to predict ______ strength, specificity, and other relevant properties based on sequence analysis.
Use bioinformatics tools to predict ______ strength, specificity, and other relevant properties based on sequence analysis.
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Conduct a comprehensive review of published literature and databases to identify ______ with documented performance in specific host systems or for similar protein expression applications.
Conduct a comprehensive review of published literature and databases to identify ______ with documented performance in specific host systems or for similar protein expression applications.
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Evaluate protein expression levels using quantitative assays such as Western blotting or ______ microscopy.
Evaluate protein expression levels using quantitative assays such as Western blotting or ______ microscopy.
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Utilize resources such as PubMed, ______ databases and scientific journals to gather relevant information.
Utilize resources such as PubMed, ______ databases and scientific journals to gather relevant information.
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Study Notes
Cloning Vectors
- pET expression vector: a type of cloning vector used for protein expression in E. coli
- Contains a T7 terminator sequence, which signals the end of transcription for T7 RNA polymerase
- Has an ampicillin resistance gene, which provides resistance to the antibiotic ampicillin and allows for selection of bacteria that have been successfully transformed with the vector
- Origin of replication from the pBR322 plasmid, which allows the plasmid to replicate within the host bacteria
- Rop (Repressor of primer) gene, which regulates plasmid copy number
- LacI (The gene coding for the lac repressor protein), which can bind to the LacO site and inhibit transcription until IPTG is added
Cloning Vectors for Eukaryotes
- Most cloning experiments are carried out with E. coli as the host, and the widest variety of cloning vectors are available for this organism
- E. coli is particularly popular when the aim of the cloning experiment is to study the basic features of molecular biology, such as gene structure and function
Gateway Cloning
- Based on the principles of recombination reactions similar to those that occur during lambda phage infection in bacteria
- Lambda phage integration and excision reactions involve recombination of attP and attB sites, creating attL and attR sites
- Gateway cloning uses a site-specific recombination system derived from bacteriophage lambda
- Two key recombination sites: attB (bacterial attachment site) and attP (phage attachment site)
- Recombination of two DNA fragments, like in the Gateway system, requires specific sequences (att) within insert and vector, which are recognized by the recombinase enzyme mixtures (Clonase)
Gateway Cloning Reactions
- Creation of entry clones: BP Reaction
- Creation of expression clones: LR Reaction
- LR Reaction takes place between the attL sites of the generated entry clone and the attR sites of the destination vector
Transcription Initiation Complex
- Open chromatin regions: increases accessibility and transcription initiation complex
- Stable promoter sequence (GC rich sequence): enhances affinity and longer residence time between promoter, TFs, and RNA pol
- Thermus thermophilus: an extremophile with a GC-rich genome, tolerating high environmental temperatures
Transcription Factor Binding Sites
- Promoters contain transcription factors binding sites
- Transcription factors: proteins that control gene expression by binding to nearby DNA
- Presence and affinity of these binding sites influence recruitment of RNA polymerase and efficiency of transcription initiation
RNA Polymerase Binding
- Promoters interact with RNA polymerase, leading to efficient recruitment and initiation of transcription
- Strong promoters have high affinity for RNA polymerase
Epigenetic Modifications and Chromatin Structure
- DNA methylation
- Histone acetylation
Regulatory Elements
- Enhancers and silencers: modulate gene expression in response to cellular signals or environmental cues
- Presence and activity of these elements can influence promoter strength by affecting the rate of transcription initiation
Promoter Selection Strategies
- Literature Review: conduct a comprehensive review of published literature and databases to identify promoters with proven performance in specific host systems
- Empirical Testing: clone the promoters into expression vectors and evaluate protein expression levels using quantitative assays
- Bioinformatics Analysis: use bioinformatics tools to predict promoter strength, specificity, and other relevant properties based on sequence analysis
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Description
This quiz covers the components of a pET expression vector, including the T7 terminator, ampicillin resistance gene, and pBR322 origin of replication. Learn about the functions of these elements in molecular biology.
