Podcast
Questions and Answers
What is the primary function of the araBAD promoter in the context of pGLO?
What is the primary function of the araBAD promoter in the context of pGLO?
Which of the following is NOT mentioned as a characteristic of a good reporter protein?
Which of the following is NOT mentioned as a characteristic of a good reporter protein?
Which reporter gene is associated with the production of light?
Which reporter gene is associated with the production of light?
In the experiment with pGLO, what method was used to visualize the expression of GFP?
In the experiment with pGLO, what method was used to visualize the expression of GFP?
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What type of reporter gene is GFP classified as in the pGLO experiment?
What type of reporter gene is GFP classified as in the pGLO experiment?
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What is the primary function of Green Fluorescent Protein (GFP)?
What is the primary function of Green Fluorescent Protein (GFP)?
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What is the role of competent E. coli cells in the pGLO experiment?
What is the role of competent E. coli cells in the pGLO experiment?
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Which of the following fluorescent proteins can be derived from Aequorea?
Which of the following fluorescent proteins can be derived from Aequorea?
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Which excitation wavelength is required to visualize the fluorescence emitted by GFP?
Which excitation wavelength is required to visualize the fluorescence emitted by GFP?
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What type of microscopy is necessary to detect GFP within living cells?
What type of microscopy is necessary to detect GFP within living cells?
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Which of the following reporter genes can be used to study gene regulation?
Which of the following reporter genes can be used to study gene regulation?
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Which system is required for detecting GFP expression in whole organisms?
Which system is required for detecting GFP expression in whole organisms?
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In the context of fusion proteins, where is the reporter gene typically placed?
In the context of fusion proteins, where is the reporter gene typically placed?
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What is a fusion protein?
What is a fusion protein?
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Which of the following organisms is mentioned as having GFP expressed in its eyes?
Which of the following organisms is mentioned as having GFP expressed in its eyes?
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What do transcriptional reporters typically include?
What do transcriptional reporters typically include?
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What is the length of the regulatory region in plasmid Ptrx-1(1kb)::GFP?
What is the length of the regulatory region in plasmid Ptrx-1(1kb)::GFP?
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Which neurons were observed to express GFP when the Ptrx-1(1kb)::GFP plasmid was microinjected into C.elegans?
Which neurons were observed to express GFP when the Ptrx-1(1kb)::GFP plasmid was microinjected into C.elegans?
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What indicates that the transgenic animals show fluorescent head neurons in the study?
What indicates that the transgenic animals show fluorescent head neurons in the study?
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What does plasmid Pssu-1(0.5kb)::GFP contain in terms of regulatory region?
What does plasmid Pssu-1(0.5kb)::GFP contain in terms of regulatory region?
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What method was used to create transgenic C.elegans with the plasmids?
What method was used to create transgenic C.elegans with the plasmids?
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What happens to GFP expression in ASJ neurons when mutations occur in the identified sequences?
What happens to GFP expression in ASJ neurons when mutations occur in the identified sequences?
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Which of the following motifs are identified as necessary for trx-1 expression in ASJ neurons?
Which of the following motifs are identified as necessary for trx-1 expression in ASJ neurons?
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What does the sequence logo image produced by the researchers represent?
What does the sequence logo image produced by the researchers represent?
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What likely conclusion can be drawn about the trx-1 regulatory region from the constructs analyzed?
What likely conclusion can be drawn about the trx-1 regulatory region from the constructs analyzed?
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How are the two identified motifs for ssu-1 expression structured?
How are the two identified motifs for ssu-1 expression structured?
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What was observed when the whole Ptrx-1(1kb)::GFP plasmid was microinjected?
What was observed when the whole Ptrx-1(1kb)::GFP plasmid was microinjected?
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What is the role of the transcription factor SPTF-1 in relation to the motifs?
What is the role of the transcription factor SPTF-1 in relation to the motifs?
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Why does the figure legend state that the figure is 'adapted' and not 'taken from'?
Why does the figure legend state that the figure is 'adapted' and not 'taken from'?
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What is a limitation of the sequence logo generated by the bioinformatics program?
What is a limitation of the sequence logo generated by the bioinformatics program?
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How many DNA motifs were compared by the researchers to produce the sequence logo?
How many DNA motifs were compared by the researchers to produce the sequence logo?
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Which region can be deleted without affecting the expression of GFP in ASJ neurons?
Which region can be deleted without affecting the expression of GFP in ASJ neurons?
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What does the deletion of the region between -103 and -180 bp indicate?
What does the deletion of the region between -103 and -180 bp indicate?
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Where is the likely regulatory region of trx-1 located?
Where is the likely regulatory region of trx-1 located?
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What effect does deleting the region between 0 and -84 bp have on GFP expression?
What effect does deleting the region between 0 and -84 bp have on GFP expression?
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Constructs 6 and 8 demonstrate that:
Constructs 6 and 8 demonstrate that:
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If the trx-1 expression in ASJ neurons is disrupted, which region is most likely implicated?
If the trx-1 expression in ASJ neurons is disrupted, which region is most likely implicated?
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What percentage of GFP expression was seen when the region between -103 and -180 bp was deleted?
What percentage of GFP expression was seen when the region between -103 and -180 bp was deleted?
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What conclusion can be drawn from the overall study regarding trx-1 regulatory sequences?
What conclusion can be drawn from the overall study regarding trx-1 regulatory sequences?
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What is the likely position of the region that controls trx-1 expression in ASJ neurons in C.elegans?
What is the likely position of the region that controls trx-1 expression in ASJ neurons in C.elegans?
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What technique was used to identify the cis-regulatory motif controlling trx-1 expression?
What technique was used to identify the cis-regulatory motif controlling trx-1 expression?
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Which nucleotides were targeted in the scanning substitution mutagenesis experiment?
Which nucleotides were targeted in the scanning substitution mutagenesis experiment?
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What does a 0% GFP expression indicate in the context of the trx-1 gene study?
What does a 0% GFP expression indicate in the context of the trx-1 gene study?
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What could be a reason for not obtaining stably transmitting lines from the trx-1 construct?
What could be a reason for not obtaining stably transmitting lines from the trx-1 construct?
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In the experiment, what does the use of orange-highlighted nucleotides signify?
In the experiment, what does the use of orange-highlighted nucleotides signify?
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What was one of the outcomes after the scanning substitution mutagenesis was performed?
What was one of the outcomes after the scanning substitution mutagenesis was performed?
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How did the researchers confirm the regions necessary for trx-1 expression?
How did the researchers confirm the regions necessary for trx-1 expression?
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Study Notes
Molecular Basis of Gene Expression - Workshop 1
- Workshop focus: Using reporter genes to study gene expression
- Course code: 5BBG0205
- Lecturer: Dr Shirley Coomber
Learning Outcomes
- Students should be able to identify different types of reporter genes
- Students should understand how reporter genes locate regulatory regions of genes
- Students should understand how transcription factors bind to specific sequences in regulatory regions of genes
Revision (Practical 4BBY1070)
- Plasmid pGLO transformed into competent E. coli cells
- Transformed cells grown on different media agar plates
- pGLO digested with restriction enzymes (practical 1)
- GFP (green fluorescent protein) cloned in front of araBAD promoter
Revision (Practical 4BBY1070 continued)
- E. coli pGLO cells grown on two LB agar plates
- One with ampicillin
- One with ampicillin and arabinose
- GFP presence in E. coli pGLO colonies detected using UV light
- Colonies glow green in UV light when grown on LB amp agar plates with arabinose
- Arabinose activates araBAD promoter, leading to GFP expression
- This is an example of a transcriptional reporter system (GFP acts as a reporter gene)
Reporter Genes
- Various genes used as reporter genes: Chloramphenicol Acetyltransferase (CAT), Luciferase, β-Galactosidase, β-Glucuronidase (GUS), Alkaline Phosphatase (AP), β-Lactamase, Fluorescent Proteins
- Good reporter proteins are easy to visualize/detect and quantifiable
Fluorescent Proteins
- Derived from Aequorea GFP or Discosoma RFP
- Excited at specific wavelengths, emitting different wavelengths
Methods for Detecting GFP within Living Cells (Slide 1)
- Fluorescence microscopy (epifluorescence or confocal) needed
- High magnification allows visualization of nucleus, mitochondria, and other cellular structures where the reporter gene is located
- Equipment needs excitation wavelength delivery and detection mechanisms
Methods for Detecting GFP within Living Cells (Slide 2)
- Whole organism imaging: Imaging system (computer/software) linked to camera with appropriate lens needed
- Example: Drosophila with GFP expressed in eyes
Making a Reporter Gene Construct
- The native gene, including core promoter and regulatory region, are upstream of the start codon
- Regulatory region and core promoter placed upstream of another reporter gene (transcriptional reporter)
- Reporter gene placed downstream from protein-coding region of native gene to create a fusion protein (translational reporter)
Definition of Fusion Protein
- Fusion proteins combine two or more proteins originally encoded by separate genes
Using GFP to Identify Regulatory Regions (Study Materials Summary)
- Study uses C. elegans
- Focuses on two genes, trx-1 and ssu-1, expressed exclusively in ASJ neurons
- Focus for this study is on trx-1
Introduction (Further Details)
- trx-1 is a gene (lower case italics)
- TRX-1 is the protein (upper case)
- Species names always in italics
- Use abbreviations for second mention of species
Introduction (Fourth Slide)
- Two plasmids used
- Ptrx-1 (1kb): 1kb region upstream of trx-1 start codon joined to GFP
- Pssu-1 (0.5kb): 500bp region upstream of ssu-1 start codon joined to GFP
Introduction (Fifth Slide)
- Whole Ptrx-1(1Kb):GFP plasmid microinjected into wild-type C. elegans to create transgenic worms
- GFP observed in ASJ neurons
- Similar results with Pssu-1(0.5Kb):GFP plasmid
Figure 4 and Questions
- Results of microinjecting plasmids with deletions of the trx-1 regulatory region
- Constructs 1 through 5 show that no regulatory control exists between positions -860 to -200
- Constructs 6 and 8 show that a regulatory region exists from -200 to -84
- Constructs 7, 9 and 10 indicate that the required regulatory region is between positions -103 and -180
Workshop Information (First Slide)
- Created a series of non-overlapping 20-30bp deletions of trx-1 promoter upstream region
- Microinjected into C. elegans
- Results shown in Figure 5
Questions Based on Figure 5
- Deletion of -173 and -200 bp upstream the trx-1 start codon prevents GFP expression in C. elegans
- The regulatory sequences for trx-1 expression in the ASJ neuron of C. elegans are located within this region of DNA.
Workshop Information (Second Slide – Continued)
- Scanning substitution mutagenesis of the region from -200 to -173
- Changes nucleotides in the Ptrx-1(1kb):: GFP plasmid
Figure 6
- Identifying the ASJ motif in the promoter of the trx-1 gene using scanning substitution mutagenesis
- DNA changes are in orange in Figure 6
- Wild-type DNA is in the top line
- Mutations in the two 6 bp sequences (separated by a 3bp linker) significantly reduce or abolish GFP expression in ASJ neurons
- CAACCC and AATTAA are likely regulatory motifs (separated by a 3bp linker)
Workshop Information (Third Slide –Continued)
- Experiments using ssu-1 promoter region
- Identified two similar motifs CTAACC and AATTAG regions located -221bp from the ssu-1 start codon
- Suggests a bipartite ASJ motif is necessary for the expression of trx-1 and ssu-1 in ASJ neurons
- Further experiments found transcriptional factor SPTF-1 binds to the ASJ motif and controls expression of trx-1 and ssu-1 in ASJ neurons of C. elegans
Additional Information
- Researchers identified and compared ASJ motifs found in 15 different C. elegans genes using a bioinformatics program (Weblogo)
- The ASJ motif is likely to be present in other C. elegans genes.
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Description
Join Dr. Shirley Coomber in this engaging workshop focused on using reporter genes to study gene expression. Learn to identify different types of reporter genes and understand their role in locating regulatory regions and transcription factor binding. Perfect for students aiming to deepen their practical understanding of genetic studies.
