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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.
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.
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.
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.
The pET expression vector is approximately ______ KB in size.
The pET expression vector is approximately ______ KB in size.
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.
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.
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.
The recombination of attP and attB sites creates ______ and attR sites.
The recombination of attP and attB sites creates ______ and attR sites.
The Gateway system involves two key ______ sites: attB and attP.
The Gateway system involves two key ______ sites: attB and attP.
The BP reaction is involved in the creation of ______ clones.
The BP reaction is involved in the creation of ______ clones.
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.
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.
Hydrogen bonding and ______ interactions occur between proteins and DNA.
Hydrogen bonding and ______ interactions occur between proteins and DNA.
Open chromatin regions have ______ accessibility to transcription initiation complex.
Open chromatin regions have ______ accessibility to transcription initiation complex.
The stable promotor sequence is characterized by a ______ rich sequence.
The stable promotor sequence is characterized by a ______ rich sequence.
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.
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 ______.
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.
Epigenetic modifications include ______ methylation and histone acetylation.
Epigenetic modifications include ______ methylation and histone acetylation.
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.
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.
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.
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.
The final growth varied due to different ______ rates under each media.
The final growth varied due to different ______ rates under each media.
TB medium showed the highest ______ rate under uninduced conditions.
TB medium showed the highest ______ rate under uninduced conditions.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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