Elucidating the p53 Signalling Network by Reverse Genetics

Elucidating the p53 Signalling Network by Reverse GeneticsReiyyan Tariq NizamiAimIn my research project I am working with four distinguishable strains Caenorhabditis elegans. I have been performing a double mutant screen using an RNAi knockout library. Cep-1 is a protein that causes apoptosis due to DNA damage in the louse. The cep-1 protein is an ortholog of the human tumor suppressor protein p53, which is found mutated in many fagcerous cells. The aim of my experiment is to find genes that are regulators of cep-1 which cause deadlyity if mutated with cep-1. Alternatively mutants which are lethal as genius mutants but survive as double mutants in combination with a cep-1 mutant are also probable genes of interest. These mutants have the potential to be regulated by drugs/proteins to change the levels of cep-1 and induce/prevent apoptosis in cells.BackgroundCancer is a disease of uncontrolled cell rea trammelg in our body. Under normal conditions a cell has regulatory proteins and check points that make real that a cell is growing at the right railyard and if for any reason the cell starts to grow irregularly and then(prenominal) these proteins stop the cell growth and cause apoptosis. Apoptosis is the programmed death of a cell, as you can imagine it is tightly regulated and red of regulation can result in catastrophe for the cell and the organism. When apoptosis is everywhere active, healthy cells will transcend and this is often seen in diseases such as neurodegenerative diseases, hematologic diseases and many other tissue damage diseases. Loss of apoptosis in turn results in cancers, autoimmune diseases and inflammatory diseases.P53 is a tumor suppressor protein that was discovered in 1979. Since then a lot of research has been done into p53 and its functions. P53 is a tumor suppressor protein that is complex in defense of the cell. It is jauntd when a cell undergoes many various kinds of stress, such as DNA damage, hypoxia, metabolic stress and oncogene activation. It is one of the more or less important barriers to cancer in many mammals. P53 works in many various parcels, primarily p53 is involved in binding to transcription factors that then activate pathways involved in cellular defense, such as preventing angiogenesis and cellular growth.Malfunctioning p53 is one of the greatest hallmarks of cancer. The majority of mutations found in p53 are due to single nucleotide substitutions in the amino acids of the DNA binding domain of the protein. Similar cancerous phenotypes are observed when either p53 loses function due to a loss of function mutation or when negative regulators of p53 are overactive or positive regulators of p53 lose their function. However p53 is quite unique amongst tumor suppressor proteins because antithetic missense mutations in the protein can cause different levels of p53 activity and hence have varying effects on the host. The effect of the mutation is further modified depending on the gene tic background of the person with the mutation.Due to the different effects of various amino acid substitutions and the varying genetic background of patients it is quite difficult to weigh large populations. This is because high- throughput sequencing and genome wide single nucleotide polymorphism maps are expensive to obtain. The prices are going down as new technologies are becoming useable however until now there have been no larger scale studies relating different mutations to varying levels of risks for different types of cancers. It is also hard to perform molecular studies on entire humans and so research is done on cell lines, mice and on cep-1 the Caenorhabditis elegans ortholog of p53.Caenorhabditis elegans is a worm in the nematodes phylum. They are a very well-studied organism due to their ease of study. C. Elegans are one of the simplest organisms that have a nervous system and that make them a very good model organism for nervous studies. They are transparent creatu res and so many molecular and cellular processes especially those involved in development have been thoroughly researched. They have a hornswoggle generation time and are very cheap to maintain. They are an extremely good model organism for performing screens because any of their nonessential genes can be knocked out easily by using RNAi.The cep-1/p53 pathway is highly conserved throughout evolution as it is an extremely important pathway that is essential for cellular survival over time.The cep-1 protein works through the followers pathway to cause apoptosis in cells suffering DNA damage.Cep-1 Egl-1/Ced-13 Ced-9 Ced-4 Ced-3 ApoptosisA similar pathway is observed in humansp53 BH3 Bcl2 Apaf1 Caspase ApoptosisA majority of these proteins are orthologs to one another. It was hoped that through my screen further proteins which interact with Cep-1 would be found and then their orthologs in humans could be found and then researched and targeted to regulate p53.Materials and Me thodsThe experiment in itself was an extremely simple but time consuming experiment. Screens were performed with mutant worm strains which were then fed RNAi, through Escherichia coli (E. coli), to silence the gene of interest and create double mutants.To begin with a molten screen is preferred over a solid screen. This is because with a liquid screen you can screen a larger sample of double mutants more easily than a solid screen. liquifiable ScreenThe following strains of bacteria and worms were utilized, E. Coli OP50, C. Elegans N2, C. Elegans GK138, C. Elegans LG12501.E. Coli OP50 Food source for C. ElegansC. Elegans N2 Wild type wormsC. Elegans GK138 Cep-1 Mutant wormsC. Elegans LG12501 Cep-1 Mutant wormsRNAi Knockout library for C. Elegans Chromosome 1Day 1 turn over worms on Nematode Growth Medium (NGM) plates with OP50 as a food source for the worms.Day 3Bleach worms that were plated on day 1 so as to only have eggs remaining on plates.Bend glass rod into L shape us ing a high temperature blaze (Bunsen burner)Pipette make white onto plates with worms on them and gently scrape the surface to mix worms and eggs with bleachPipette fluid into Eppendorf tube and spin at sludge speed for 3 minutes take away most of the fluid and keep as much of the pellet as possibleSuspend pellet with bleachRepeat steps 3 and 4Suspend pellet using M9 firmness of purpose centrifugate at high speed for 1 minuteAspirate most of the fluid and keep as much of the pellet as possibleRepeat 7 and 8Suspend pellet using M9 solution and vortex Eppendorf tube at low speed to mix solutionPlace Eppendorf tubes in slow rocker in a 20 fridge overnight to allow eggs to hatchDay 4Replicate bacteria containing RNAi from knockout library using a sterile 96 pin replication tool into a 96 well containing Liquid Broth (LB) with Ampicillin and allow the bacteria to replicate overnight at 37C in an brooderInduce transcription of RNAi using adding 0.1 Molar IPTG into swell and postal service in a shaker for 1 hour.Pellet bacteria by centrifuging in a cold centrifuge at 5C for 5 minutes at 2,500 gRemove the supernatant by flipping over the well quickly but carefully so as to keep pelleted bacteria in the wellsSuspend bacterial pellet in wells using NGMPipette worms into wells and place in 37C shakerDay 8Remove worms from shaker gently making sure not to tilt the 96 well platesObserve and record phenotype of the worms correspond phenotype mingled with the 3 different strains of wormsLeave worms in 20C fridge overnightDay 9Remove worms from fridge gently making sure not to tilt the 96 well platesObserve and record phenotype of the wormsCompare phenotype from previous dayCompare phenotype between the 3 different strains of wormsSequencing afterward potential hits were found in the liquid screen the RNAi from these bacteria were epochd to ensure that the sequence of the RNAi was correct and hadnt randomly mutated over time. RNAi was prepared by using a Qiagen mini prep spin kit.Suspend bacteria in 250 l Buffer P1 and place in a microcentrifuge tubeMix 250 l Buffer P2 and shake the miscellanea by flipping tube over a few timesTo the mixture add 350 l N3 buffer, mix well quicklyCentrifuge at 13000 rpm for ten minutesAspirate supernatant into new tubeCentrifuge again for a roughly 1 minute and discard the flow throughWash spin column with 0.5 ml PB buffer and centrifuge for 1 minute, discard flow throughWash spin column with 0.75 ml PE buffer and centrifuge for 1 minuteDiscard flow through and centrifuge at maximum speed for 1 minutePlace prep column in a sterilized 1.5 ml microcentrifuge tube take 50 l of water to prep spin column and allow to rest for 1 minute after which centrifuge for 1 minuteAfter the Qiagen miniprep is complete the tubes were sent for sequencing to The Centre for Applied Genomics where it was sequenced and results were obtained within a week.Solid ScreenSolid screens were performed on genes which were found to have increa sed lethality with cep-1 deletion or increased survivability with cep-1 deletion.The following strains of bacteria and worms were utilized, E. coli OP50, C. Elegans N2, C. Elegans TG12E. Coli OP50 Food source for C. ElegansC. Elegans N2 Wild type wormsC. Elegans TG12 Cep-1 Fluorescent tagged (GFP) wormsC. Elegans Ned-8 Positive controlC. Elegans HT115 Negative controlRNAi Knockout library for C. Elegans Chromosome 1Day 1)Streak RNAi bacteria of interest from RNAi Library to obtain single colonies. Allow them to grow overnight in 37C incubatorDay 2)Pick and grow a single colony in 5 ml of LB + Amp + Tet overnight in a 37C shakerDay 3)Add 0.1M IPTG for 4 hours to induce RNAiPlate 100 l of induced bacteria on RNAi plates incubate at 37C incubator overnightDay 4)Pick 5 worms at the same stage and plate onto RNAi platesAllow to grow over 4 age at 20CDay 8)Score phenotypes and compared between different strainsDay 9)Score phenotypes again on next day and compare between differen t strains and the previous dayFluorescent MicroscopyWe used a Differential interference contrast (DIC) microscopy to observe localization of cep-1 in TG12 worms. Worms were mounted using the following methodPlace a drop or two of hot liquid agarose onto microscope slideImmediately place a second slide perpendicularly on top of the agaroseAllow agarose to settle for one to three minutesGently slide off second slide so as to go out an agarose patch behindAdd a drop of 1mM Levamisole to paralyze worms and prevent their movementPick worms and place them on the slideSlowly place coverslip on top of worms, be very gentleOnce prepared the slides were observed using a DIC microscope to find any irregularities in the localization or occur of cep-1 in the worms, especially in the germline and the eggs.ResultsThe aim of this experiment was to find genes that cause lethality as double mutants with cep-1 mutants but not in wild type worms, these genes would be positive regulators of cep-1 and cause apoptosis through cep-1. The screen was also designed to find worms that cause lethality in wild type worms but not in cep-1 mutants, these would be negative regulators of cep-1 and cause apoptosis through cep-1.The results were gathered and tabulated to allow for an easier and better analysis of data.Note There is a lot of data and so only data that is relevant is shown.Liquid Screen ResultsLegendL = Synthetic LethalE = Embryonic LethalG = Slow GrowthB = Egg laying abnormal R = No RNAi C = Contamination A = Larval Arrest S = Sterile V = Variable Morphology P = Lethal progeny W = No WormsCells of interest are highlightedChromosome 1 Plate 1N21357911131517192123ARGG/LLCA/CA/CERE/SGRRIS/LSRRRRKRRRRMRRRRSORRS/ERcep-1 (gk)1357911131517192123ARLC/LC/LG/ECG/SG/LC/LERE/SGRRIE/SE/SRRRRKRRRRMRG/ERRR/G/SORRSRcep-1 (lg)1357911131517192123ARC/LLLE/PCG/LGLERE/SGRRIS/BS/BRRRRKRRRRMRE/GRRRSORRS/ERChromosome 1 Plate 5N224681012141618202224ACEGEIEKRMRRROGRcep-1 (gk)24681012141618202224ACEGI EKRMRRRORcep-1 (lg)24681012141618202224ACEGIEKRMRRRORKey results of interest in liquid screenLethality in gk and lg but not in N2WellGeneN2gklgA23F53G12.5G/EE/PM03Y95B8A_85.hG/EE/GLethality in N2 but not in gk or lgWellGeneN2gklgG04F25H2.10ESolid Screen Results3 hits of interest were obtained from all the plates that were screened. These 3 hits were then repeated multiple times on solid media to make sure the results were accurate.N2lggkA23 -1A23 -2A23 -3N2lggkM03 -1EM03 -2EM03 -3N2lggkG04 -1LAG04 -2EG04 -3The M03 strain showed opposite results in the solid screen in comparison to the liquid screen. However since the results were consistent we decided to study both M03 and G04 under the microscope with fluorescent markers.Fluorescent microscopy resultsNo major mislocalization of GFP tagged cep-1 was found and no extra or lack of GFP tagged cep-1 was observe