Protein Ratio Control: Promoters and RBSs

Play an AI-generated podcast conversation about this lesson

Why is coordinated transcription an advantage of using the same promoter for two genes in controlling protein ratios?

It ensures both genes are expressed together at a fixed ratio, simplifying the control and predictability of protein production.

What is a limitation of controlling protein ratios using the same promoter but different RBS strengths?

It is difficult to dynamically regulate the individual levels of each protein independently, as both genes are transcribed together.

How does using different promoters to control protein ratios provide more flexibility compared to using the same promoter?

Different promoters allow independent control of transcriptional levels based on promoter strength and inducibility, whereas the same promoter links transcription of both genes.

What is a potential drawback of placing two strong promoters closely together to control protein ratios?

There is a possible risk of transcriptional interference, where one promoter affects the activity of the other, leading to unpredictable expression levels. Signup and view all the answers

In scenarios where precise control of protein stoichiometry is needed, such as in multi-enzyme pathways, why is using different promoters and different RBSs advantageous?

This strategy allows maximum flexibility in tuning protein expression by independently adjusting both transcription and translation levels. Signup and view all the answers

What makes optimizing protein expression more complex when using different promoters and different RBSs, compared to other strategies?

Both transcription and translation levels must be individually fine-tuned, increasing the complexity of balancing protein expression. Signup and view all the answers

Why might controlling protein ratios with different promoters and different RBSs lead to higher variability in protein expression?

Due to the combinatorial effects of different promoters and RBSs, leading to more factors that can influence protein production. Signup and view all the answers

How can using different promoters and different RBSs to control protein ratios burden the cell?

It can increase the metabolic demand for transcription and translation, as each gene's expression is independently driven, potentially overtaxing cellular resources. Signup and view all the answers

How does the mechanism of action of bacterial anti-sense RNAs differ from that of eukaryotic siRNAs in gene regulation?

Bacterial anti-sense RNAs often physically block translation or mRNA stability, while eukaryotic siRNAs induce degradation of complementary mRNAs. Signup and view all the answers

Describe how miRNAs regulate gene expression, and explain why this regulation is considered post-transcriptional.

miRNAs regulate gene expression by binding to target mRNAs, often in the 3' UTR, leading to translational inhibition or mRNA destabilization. This is post-transcriptional because it occurs after the mRNA has already been transcribed. Signup and view all the answers

Explain how anti-sense RNA can prevent the formation of an inhibitory structure within a target mRNA.?

Anti-sense RNA binds to regions of the mRNA that would normally pair internally to form an inhibitory structure, preventing this structure from forming. Signup and view all the answers

How does the function of siRNAs within the RISC lead to gene silencing?

siRNAs guide the RISC to mRNAs with perfect complementarity, leading to the cleavage and degradation of the mRNA, thus silencing the gene. Signup and view all the answers

Describe the role of the Signal Recognition Particle (SRP) in protein export.?

The SRP recognizes a signal peptide on the emerging protein, pauses translation, and guides the ribosome-protein complex to the cellular membrane for export. Signup and view all the answers

What is the significance of FtsY in prokaryotic protein export, and what is its eukaryotic counterpart?

In prokaryotes, FtsY is the SRP receptor that the SRP-ribosome complex binds to. The eukaryotic counterpart of FtsY is SR (SRP receptor). Signup and view all the answers

Explain how anti-sense RNA binding can lead to endonuclease-mediated cleavage of mRNA.

Anti-sense RNA forms a duplex with the target mRNA, which then becomes a substrate for endonucleases that cleave and degrade the mRNA. Signup and view all the answers

Describe how anti-sense RNAs can cause exclusion of protein binding from mRNA?

Anti-sense RNAs can form duplexes with regions of the target mRNA, thus blocking these proteins from binding and affecting gene expression. Signup and view all the answers

What is the function of the protective proteins that coat T-DNA during Agrobacterium infection?

The proteins protect the T-DNA from degradation by plant nucleases. Signup and view all the answers

Describe the mechanism by which T-DNA integrates into the plant genome. What repair pathway is utilized?

T-DNA integrates randomly into the plant genome via the plant's double-strand break repair mechanism. Signup and view all the answers

What are opines, and what role do they play in the Agrobacterium infection process?

Opines are unique nitrogen-carbon sources synthesized by crown gall tumors due to T-DNA's opine biosynthesis genes; they are metabolized exclusively by Agrobacterium. Signup and view all the answers

How have scientists adapted the Ti-plasmid for plant genetic engineering, and what modifications are made?

Scientists remove tumor-inducing genes and insert desired transgenes into the T-DNA region of the Ti-plasmid. Signup and view all the answers

Explain one advantage and one disadvantage of transgene expression in the chloroplasts of plants, compared to nuclear expression.

Advantage: higher yields, stable integration, and maternal inheritance. / Disadvantage: overcoming heteroplasmy. Signup and view all the answers

Briefly describe how T-DNA is transferred from Agrobacterium into the plant cell.

T-DNA is transferred via a Sec-dependent export system into the plant cell nucleus. Signup and view all the answers

What is the primary mechanism of DNA repair in SDN1, and what type of mutations does it typically introduce?

Non-homologous end joining (NHEJ), which introduces random mutations (insertions/deletions). Signup and view all the answers

In SDN2 genome editing, what is required in addition to a double-strand break (DSB) to achieve precise gene modification, and what repair mechanism is utilized?

A short DNA repair template carrying specific mutations is required; homology-directed repair (HDR) is utilized. Signup and view all the answers

Explain the key difference in functionality between a shuttle plasmid and a suicide vector in genetic engineering.

A shuttle plasmid can replicate in multiple host organisms, while a suicide vector can only replicate in the initial host and is designed to integrate into the genome of the destination organism without autonomous replication. Signup and view all the answers

List the two essential elements of a shuttle plasmid, and explain why each is necessary for its function.

<ol> <li>Origins of replication are required for replication in both organisms. 2. Different marker genes are required for selection in both organisms.</li> </ol> Signup and view all the answers

A researcher wants to clone a gene into the pET28(+) vector such that the expressed protein has a His6-tag at the N-terminus. Describe the key features of the forward primer that would be needed for PCR amplification of the gene for restriction-based cloning using the NdeI restriction site.

The forward primer should include an NdeI recognition site (CATATG) and a sequence that ensures the start codon (ATG) is in frame with the His6-tag. It should also have enough bases to allow for proper binding to the DNA. Signup and view all the answers

Describe the required modification to the reverse primer to ensure cloning a gene into pET28(+) results in a protein with a C-terminal His6-tag, using the XhoI restriction site.

The reverse primer should exclude a stop codon and include an XhoI restriction site. This ensures the protein continues to be translated into the His6-tag sequence on the plasmid. Signup and view all the answers

Describe the primer design strategy required to clone a gene into pET28(+) at the NcoI and XhoI sites to omit any His-tag.

The forward primer should incorporate an NcoI site, while the reverse primer should include an XhoI site and a stop codon. The sequences must also allow for in-frame cloning. Signup and view all the answers

The pET vector has a copy number of 7. If pET22a (with AmpR) and pET28b (with KanR) are simultaneously transformed into cells and grown on Amp/Kan plates, what general outcome would you expect regarding the plasmid copy numbers in the cells relative to if they were transformed separately, and why?

The plasmids will likely compete for replication machinery, resulting in variable copy number ratios in each cell and potentially lower average copy numbers for each plasmid compared to individual transformations. However, the antibiotic selection maintains both plasmids. Signup and view all the answers

A researcher is using a suicide vector to insert a gene into a bacterial chromosome via homologous recombination. What crucial element is missing from the suicide vector that prevents its replication within the target bacteria, and why is this necessary for the intended outcome?

The suicide vector lacks a functional origin of replication (ori) for the target bacteria. This prevents the plasmid from replicating autonomously, forcing it to integrate into the chromosome via homologous recombination to be maintained. Without integration, it gets lost. Signup and view all the answers

Where does SRP release and ribosome translation resume, and in what type of cells does this occur?

SRP releases and ribosome translation resumes at the Sec translocon. This occurs at the rough endoplasmic reticulum in eukaryotes and the plasma membrane in prokaryotes. Signup and view all the answers

Explain the key difference in the state (folded or unfolded) of proteins transported by the Sec and Tat pathways, and indicate which system requires ATP.

The Sec system transports proteins in an unfolded state using ATP. The Tat pathway transports fully folded proteins and does not require ATP. Signup and view all the answers

Explain why using different antibiotic resistance markers (e.g., AmpR and KanR) on two compatible plasmids (like pET22a and pET28b) can be beneficial when co-transforming them into E. coli.

Different antibiotic markers allow for selection of cells containing both plasmids. The presence of both antibiotics, ampicillin and kanamycin, ensures that only cells harboring both plasmids survive, due to the individual resistance genes they carry. Signup and view all the answers

What is the role of signal peptidase, and in which protein transport system is it utilized?

Signal peptidase cleaves off the signal peptide after translocation in the Sec protein transport system. Signup and view all the answers

Describe the twin-arginine motif and its importance in protein transport.

The twin-arginine motif is a signal peptide recognized by the Tat pathway for transporting fully folded proteins across the membrane. Signup and view all the answers

Define codon wobbling and explain its significance in translation.

Codon wobbling is the flexibility in base pairing between the third position of a codon on mRNA and the corresponding anticodon on tRNA. This allows a single tRNA to recognize multiple codons coding for the same amino acid. Signup and view all the answers

Summarize the concept behind the student's investigation. What is she trying to optimise?

The student investigates three different strategies for the co-expression of the genes of two proteins to find the optimal ratio of both proteins in cell-free extracts Signup and view all the answers

Predict the effect of using the same promoter but different RBS (ribosome binding site) sequences for two co-expressed genes on their protein production levels.

Using the same promoter but different RBS sequences will cause variations in translational efficiency, as some ribosomes will bind more efficiently than others. This will result in variable protein ratios. Signup and view all the answers

Outline one potential problem that might arise from using different promoters but the same RBS for the co-expression of two proteins.

Using different promoters can lead to temporal expression differences, where one protein is expressed earlier or more strongly than the other. If the RBS is identical, the protein produced first might occupy most of the ribosomes, limiting the translation of the second protein. Signup and view all the answers

Describe the crucial components of a knockout plasmid that are essential for achieving targeted gene replacement in yeast.

A selectable marker, flanking homologous regions, and a terminator sequence are the crucial components. Signup and view all the answers

What is the function of guide RNA (gRNA) in CRISPR-Cas9-based gene inactivation, and why is it important for precise gene editing?

The gRNA directs the Cas9 nuclease to a specific DNA sequence for targeted double-strand breaks, enabling precise gene editing. Signup and view all the answers

When overexpressing a heterologous protein, what is the role of a strong promoter in an expression vector, and provide an example of a commonly used yeast promoter?

A strong promoter drives high-level transcription of the heterologous gene. Examples are TEF1, GAL1, and PGK1. Signup and view all the answers

Why is a yeast origin of replication (2µ or CEN/ARS) necessary in an expression vector used for heterologous protein overexpression?

Yeast origin of replication is necessary for stable maintenance and replication of the expression vector inside the yeast cell. Signup and view all the answers

What bacterial elements are required in a yeast expression vector, and what purpose do they serve?

A bacterial origin of replication and an antibiotic resistance marker are required for vector amplification and selection in E. coli. Signup and view all the answers

Describe two common methods used for transforming yeast cells with DNA, and briefly explain the principle behind each method.

LiAc-PEG uses heat shock to facilitate DNA uptake, and electroporation uses an electric field to create temporary pores in the cell membrane, allowing DNA to enter. Signup and view all the answers

Why is it important to determine the ploidy of a yeast strain before attempting gene inactivation?

Diploid strains require inactivation of both copies of a gene for complete knockout, whereas haploid strains only require one. Signup and view all the answers

Besides antibiotic resistance, what is another type of selectable marker that can be used in yeast, and how does it work?

Auxotrophic markers (e.g., HIS3, LEU2) can be used. They complement metabolic deficiencies in the host strain, allowing only transformed cells to grow in specific media. Signup and view all the answers

Shuttle Plasmid

Transfers a plasmid between different species. Cloned and amplified in E.coli, then transferred to the host organism.

Suicide Vector

Lacks the ability to replicate in the destination host. Integrates into the genome, then the plasmid is lost. Used to insert genes without maintaining the plasmid.

Shuttle Plasmid Essentials

Origin of replication for both organisms.
Different marker genes for both organisms.

pET28(+) Vector Feature

The pET28(+) vector allows ATG start codon insertion via NdeI or NcoI restriction sites, enabling in-frame cloning.