5BBG0205 Molecular Basis of Gene Expression Workshop (2024-2025) Answers PDF

5BBG0205 Molecular Basis of Gene Expression Workshop. Applications of RNAi ANSWERS Dr Shirley Coomber With Polleverywhere Learning outcomes After this workshop students will Understand what is meant by the term C. elegans RNAi feeding library. Understand the Timmons...

5BBG0205 Molecular Basis of Gene Expression Workshop. Applications of RNAi ANSWERS Dr Shirley Coomber With Polleverywhere Learning outcomes After this workshop students will Understand what is meant by the term C. elegans RNAi feeding library. Understand the Timmons and Fire feeding vector (L4440) and how it produces double stranded RNA molecules in living E. coli cells. Understand how C. elegans RNAi feeding experiments are used to help identify gene function. Understand how different gene constructs are expressed in E. coli and/or C. elegans. Useful information sources 5BBG0205 Lecture: RNA mediated Regulation of Gene Expression (RNA interference) 5BBG0205 Workshop 1: Workshop 1: Promoter mapping using reporter genes González-Barrios, M., Fierro-González, J.C., Krpelanova, E., Mora-Lorca, J.A., Pedrajas, J.R., Peñate, X., Chavez, S., Swoboda, P., Jansen, G. and Miranda-Vizuete, A. (2015) Cis- and trans-regulatory mechanisms of gene expression in the ASJ sensory neurons of Caenorhabditis elegans. Genetics. 200:123-34. https://doi.org/10.1534/genetics.115.176172 Boutros, M., Ahringer, J. (2008) The art and design of genetic screens: RNA interference. Nat Rev Genet 9, 554–566. https://doi.org/10.1038/nrg2364 Kim, S. (2001) http://C.Elegans: Mining the functional genomic landscape. Nature Reviews Genetics 2, 681–689. https://doi.org/10.1038/35088523 Ahringer, J., ed. Reverse genetics (April 6, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.47.1 , http://www.wormbook.org. http://wormbook.org/chapters/www_introreversegenetics/introreversegenetics.html Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., & Ahringer, J. (2000). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature, 408(6810), 325–330. https://doi.org/10.1038/35042517 Part A. Reverse genetics based on RNAi Part A Introduction (slide 1) RNAi based methods have been developed to knock down gene expression of different gene targets in a variety of organisms. Figure 2 is a simple diagram showing different RNAi methods that can be used to knock down gene expression of native target genes. On next slide Figure 2. Overview of RNAi screening approaches used in different organisms. A. Long double stranded (ds) RNAs for the target gene are introduced into Caenorhabditis elegans. B. Drosophila cells are bathed in double stranded RNA. C. siRNAs for the target gene can be introduced into human (or vertebrate) cells by transfection. If dsRNA is used the native Dicer cuts it to produce siRNA which is loaded onto native RISC. If siRNA is used this can be loaded directly onto native RISC. In all cases the RISC/guide strand complex can then silence/knock down the expression of the target gene. Taken from Boutros and Ahringer (2008). Figure 2. Overview of RNAi screening approaches used in different organisms.  Question 1. What is reverse Reverse genetics genetics? Forward (classic) genetics DNA/protein sequence for gene known from whole Start with mutant (phenotype) organism genome sequence dentify mutant allele (genome location) Bioinformatics used work out what type/function of protein is likely to be by similarity of DNA sequence gene protein sequences of functionally known proteins. Mutation of gene or gene Deduce protein sequence knock down (or other) used to from DNA sequence create a ‘mutant’ phenotype that identifies specific protein function Use bioinformatics to try and work out what type of protein Specific function of protein in and relate most likely protein organism now confirmed by type/function to mutant ‘wet’ experimental results  Question 2. What do you think is the main limiting factor in using RNAi to deliberately knock down expression of a native gene? Delivery of RNA into target cell/tissue/whole organism Main problem is how do you get the double stranded RNA or siRNA into the living cell to see the RNAi effect Next slide Figure 3 shows how double stranded RNA can be introduced into C. elegans in three different ways to knock down gene expression. Figure 3. Methods used to introduce double stranded RNA into C. elegans. A. RNAi by feeding. B. RNAi by injection. C. RNAi by soaking. Adapted from Kim, S. (2001). Question 3. Which one of the three ways to introduce double stranded RNA to C. elegans is technically the easiest? RNAi by feeding Why? Purifying good quality double stranded RNA for injection or soaking methods is hard (due to RNases everywhere that break down RNA). Microinjection very skilled and time consuming. Question 4. Which one of the three ways to introduce double stranded RNA to C. elegans is technically the hardest? RNAi by injection Why? Purifying good quality double stranded RNA for injection or soaking methods is hard (due to RNases everywhere). Microinjection very skilled and time consuming. Part B. The Ahringer C. elegans RNAi feeding library Part B introduction (slide 1) The RNAi feeding library for C. elegans was created by Prof. Julie Ahringer’s research group at the Wellcome CRC Institute, University of Cambridge. The RNAi feeding library currently has 21,529 different clones, made by cloning gene-specific genomic fragments between two inverted T7 promoters in a plasmid called L4440 (Fraser et al., 2000). The library contains different clones which target around 90% of identified C. elegans genes. The Escherichia coli strain HT115 strain must be used as a host for the plasmid L4440 RNAi clones. The E. coli strain HT115 genome contains an IPTG inducible T7 polymerase gene and has a disruption of it’s native RNase III gene (a dsRNAse) caused by the insertion of transposon Tn10 which carries a tetracycline Part B introduction (slide 2) The Ahringer RNAi feeding library has be subdivided into subsets for Chromosome I, Chromosome II, Chromosome III, Chromosome IV, Chromosome V, Chromosome X, Chromatin, Transcription Factors and Phosphatase. González-Barrios et al. (2015) used the transcription factor subset for their experiment to identify the transcription factor regulating trx-1 and ssu-1 in ASJ neurons. Figure 4 shows the vector used to create the Ahringer RNAi feeding library. Figure 4. Two plasmid maps of the Timmons and Fire feeding vector (L4440) from Addgene. The bottom image was created by taking the L4440 plasmid DNA sequence from Addgene and producing a plasmid map using Snapgene (Slide 1). Figure 4. Two plasmid maps of the Timmons and Fire feeding vector (L4440) from Addgene. The bottom image was created by taking the L4440 plasmid DNA sequence from Addgene and producing a plasmid map using Snapgene (Slide 2).  Question 5. How big is plasmid L4440 (without adding any inserts)? 2790 bp or 2.79 kb Question 6. What antibiotics resistance marker gene(s) does plasmid L4440 carry? AmpR = ampicillin resistance (used to select for presence of plasmid in E. coli) Question 7. Where are DNA inserts put into plasmid L4440? In the MCS = multiple cloning site which has a number unique (cut once in plasmid) restriction enzyme sites  Question 8. What origin of plasmid replication (ori) is present on plasmid L4440? The Snapgene map is identifying pBR322 ori However Google – ‘pBR322 origin of replication’ You find https://en.wikipedia.org/wiki/PBR322 https://blog.addgene.org/plasmid-101-origin-of-replicatio n L4440 (and pBR322) ori is originally from plasmid pMB1 – many E. coli plasmids used in molecular biology have this origin of replication but mutated versions of original that give have a higher plasmid copy number/cell. Typically 300-500 copies of plasmid per cell. Question 9. What organism(s) can the plasmid L4440 replicate in? E. coli Origin of replication only works in E. coli and coli and some other species of bacteria. Plasmid L4440 can not replicate in C. elegans using the prokaryote origin of replication. Question 10. What organism(s) can plasmid L4440 express it’s genes in? E. coli The prokaryote promoters on plasmid L4440 only work in E. coli and some other species of bacteria. The genes on L4440 are not expressed in C. elegans. Question 11. Once a plasmid L4440 containing a DNA insert is introduced into competent E. coli strain HT115 cells how is the gene carried by the insert expressed to produce RNA in living E. coli cells? Add IPTG From introduction The E. coli strain HT115 genome contains an IPTG inducible T7 RNA polymerase gene. N.B. as it is ‘IPTG inducible’ this must mean the T7 RNA polymerase gene is being expressed by a lac operon promoter. If you add IPTG to the media T7 RNA polymerase will be produced and the DNA insert between the T7 promoters will be transcribed into RNA. Addition information on E. coli genotypes The full genotype of E. coli strain HT115 is as follows: F-, mcrA, mcrB, IN(rrnD-rrnE)1, lambda -, rnc14::Tn10(DE3 lysogen: lavUV5 promoter -T7 RNA polymerase) (IPTG-inducible T7 RNA polymerase) (RNase III minus). If you want to know what all this genotype information means see https://openwetware.org/wiki/E._coli_genotypes Google ‘lavUV5 promoter’ https://en.wikipedia.org/wiki/LacUV5 The lacUV5 promoter is a mutated promoter from the Escherichia coli lac operon which is used in molecular biology to drive gene expression on a plasmid. lacUV5 is very similar to the classical lac promoter, containing just 2 base pair mutations in the -10 hexamer region, compared to the lac promoter.  Question 12. When the gene carried by the insert in L4440 is expressed what type of the RNA product is made? Double stranded RNA molecules Promoters have direction that is based on their DNA sequence Left side T7 promoter and transcription This is also happening on the other side of the insert but the orientation of the T7 promoter means the other DNA strand is used as the template for transcription. Question 13. You have transformed plasmid L4440 containing a DNA insert into competent cells of E. coli strain HT115. What would you add to the LB agar plates to select for colonies of E. coli that contain the plasmid and why? Ampicillin (50 µg/ml final concentration) to select for E. coli contain the plasmid L4440 containing a DNA insert. Tetracycline (probably 15 µg/ml final concentration) to select for E. coli strain HT115 that still have transposon Tn10 insertion in E. coli rnc14 gene (= RNAse III minus). From Part B introduction The E. coli strain HT115 genome contains an IPTG inducible T7 RNA polymerase gene and has a disruption of it’s native RNAse III gene (a dsRNAse) caused by the insertion of transposon Tn10 which carries a tetracycline resistance gene.  Question 14. Why is E. coli strain HT115 RNase III minus? From RNAi lecture E. coli RNase III is a class 1 member of the RNase III family Related to Dicer and Drosha in terms of function. So can cut double stranded RNA E. coli strain HT115 is RNase III minus (produces no RNase III) Therefore double stranded RNA produced from plasmid L4440 will not be cut into (useless) multiple pieces in E. coli by its native RNase III as it is made. Workshop information Nematode Growth Medium (NGM) agar plates are is typically used in a C. elegans RNAi feeding experiment using the clones in Ahringer RNAi feeding library. NGM agar contains 3 g of NaCl, 2.5 g of peptone and 20 g of agar per litre. Question 15. What else needs to be added to the NGM agar media plates before the plates are used in a C. elegans RNAi feeding experiment? Ampicillin (though actually use carbenicillin) – to maintain selection for E. coli that contain plasmid L4440 IPTG – to induce expression of T7 RNA polymerase from lac operon promoter so that T7 RNA polymerase can produce double stranded RNA product. NOTE. It would be a waste of E. coli ‘energy’ to make the double stranded RNA until it was needed in experiment. E. coli trying to make lots of a RNA (or protein) product will divide more slowly because they struggling to take in enough nutrients required from the environment even in a rich media. Carbenicillin? Ampicillin? The β-lactamase (bla) gene (often called AmpR on plasmid maps) confers resistance to ampicillin and carbenicillin (a semi-synthetic ampicillin analog). Carbenicillin is sometimes chosen because it is more stable than ampicillin (less likely to break down in agar plates used for days). Carbenicillin Ampicillin Question 16. The NGM agar media plates are spread with E. coli strain HT115 cells containing the plasmid L4440 with gene specific DNA insert. What type/form of C. elegans are then typically put on the surface of these agar plates? L4 or L3 stage hermaphrodite Part C. Identification of a transcription factor that regulates trx-1 and ssu-1 expression in ASJ neurons using the Ahringer C. elegans RNAi feeding library Part C Introduction (slide 1) In González-Barrios et al. (2015) used two stable strains of C. elegans called Ptrx-1::gfp and Pssu-1::gfp to performed a feeding RNAi screen using the transcription factor sub- set of the Ahringer C. elegans RNAi feeding library. Note. A stable line has a DNA construct e.g. Ptrx-1::gfp integrated into the C. elegans genome. The Ptrx-1::gfp construct integrated into the C. elegans genome is the promoter Ptrx-1 controlling the expression of GFP. The Pssu-1::gfp integrated into the C. elegans genome is the promoter Ptrx-1 controlling the expression of GFP. Each plasmid in this library was known to knock down the expression of a different transcription factor by expressing a double stranded RNA molecule that targets the mRNA of that transcription factor using the RNA interference mechanism. In total, 403 RNAi clones (plasmids) each targeting a different transcription factor, where used. The researchers independently scored each RNAi plasmid  Question 17. How many transcription factors were identified using the C. elegans RNAi feeding experiment in González-Barrios et al. (2015)? What is/are the transcription factor(s) name(s)? One transcription factor called SPTF-1 Question 18. Draw a flow diagram to explain how the double stranded RNA molecule produced by E. coli containing plasmid L4440 with a specific gene insert knocks down gene expression in living whole C. elegans worms? E. coli containing plasmid L4440 with a sptf-1 gene insert in the presence of IPTG will produce lots of double stranded RNA Whole E. coli are eaten by stable C. elegans stain Ptrx-1::gfp (as food) somethings missing here! Double stranded RNA is cut within C. elegans cells to siRNA by Dicer Guide strand of siRNA binds to RISC which then stops the native sptf-1 mRNA producing transcription factor SPTF-1 C. elegans stain Ptrx-1::gfp no GFP produced in ASJ neurons Therefore transcription factor SPTF-1 is required to switch on trx-1 expression in ASJ neurons  Somethings missing here! How does double stranded RNA get from being inside an E. coli cell to inside a C. elegans cell? oli cells are digested (broken open) in the worms gut and dsRNA is relea dsRNA can also cross cell membranes into cells. C. elegans proteins SID-1 and SID-2 encode proteins that form dsRNA specific trans-membrane channels that assist dsRNA uptake into C. elegans cells. dsRNA can also move through the fluid filled body cavity to all parts of the worm. So dsRNA introduced by feeding, soaking or injection methods can move into all cells of the C. elegans body.