AbstractThe model organism Caenorhabditis elegans was used in this experiment to test whether or not certain experimental genes were involved in chemotaxis. The Caenorhabditis elegans were fed Escherichia coli containing RNAi that would knock down the experimental gene and allowed to reproduce for a week so that a chemotaxis assay could be run to test if the experimental genes were affected. The chemotaxis indexes were calculated and the experimental genes were compared negative control where no significant difference was observed. The chemotaxis indexes for the positive and negative controls were not very robust, so the experimentals cannot be compared to the controls, resulting in an inability to accurately accept or reject the null hypothesis and to accurately determine whether or not the genes ref-2 and T26C5.2 are involved in chemotaxis. IntroductionCaenorhabditis elegans (C. elegans) is a model organism used in various research studies. C. elegans are ideal for this experiment because the worms have a well known genetic background and the replicate quickly with little maintenance (Anderson et al. 2008). In order to eat, C. elegans relies on chemotaxis, the process of moving towards food by attraction to a chemical. C. elegans moves towards attractive scents and away from unattractive scents using cilia. Chemotaxis in C. elegans is regulated by a neuron system (UCSB 2017). Neurons AWC and AWA are involved in chemo-attraction while AWB is involved in chemo-repulsion. The molecule’s sensory neurons, AWA, AWC, and AWB are located on cilia and the cilia contain receptors for odorant molecules (UCSB 2017). Information is transferred from the sensory neurons to motor neurons, which dictate whether the molecule will move towards the odorant or away from it (UCSB 2017). The knock-down (reduction) of gene expression is a common method in determining protein function in C. elegans, “the worm”. This process uses RNAi (RNA interference) to reduce the expression of certain proteins. This method allows researchers to determine the function of the gene that is knocked down by the RNAi (Maeda et al. 2001). Double stranded RNA, or dsRNA injected into adult worms greatly interferes with the function of the gene the dsRNA targets (Fire et al. 1998). The use of this process led to the conclusion that the ODR-10 gene in C. elegans is directly involved in chemotaxis involving diacetyl (Zhang et al. 1997). The purpose of this experiment is to determine if the C. elegans genes T26C5.2 and ref-2 are involved in the worm’s chemotaxis. ref-2 is a gene known to be required to initiate the differentiation of AIY, a neuron that is triggered by acetylcholine (WormBase 2017). T26C5.2 is a gene involved in protein coding, and is known to be enriched in the PVD and OLL neurons (WormBase 2017). I believe that the genes T26C5.2 and ref-2 are involved in the ability of C. elegans to respond to chemicals in the environment, and therefore the worms with the knocked down genes will not move towards the diacetyl in the chemotaxis assay. MethodsThis study was conducted in the labs in the Life Science Building at University of California Santa Barbara. It was a part of the LURE program, the Large-scale Undergraduate Research Experience. Setting Up the RNAi Plates:I labeled four RNAi plates with numbers identifying the RNAi used for each plate, including T26C5.2, ref-2, and the positive (ODR-10) and negative (L4440) controls. ODR-10 is a gene that is known to be involved in chemotaxis, so we will expect to see a changed chemotaxis assay for the worms. The negative control is expected to show a normal chemotaxis assay, so the controls let us compare our experimental results to results that we know are correct. Each plate contained Escherichia coli with the RNAi corresponding to the desired gene for knock-down. I transferred 3-8 worms to each RNAi plate from a homogenous worm solution. I counted how many worms were on each plate using a dissecting microscope and recorded the data. I then sealed the plates and taped them together to incubate for 7 days at 15 degrees Celsius (UCSB 2017).Chemotaxis Plate:I set up four chemotaxis plates using four large agar plates. I drew a line down the center of each plate and drew a dime-sized circle in the center of each plate, with two smaller circles on the edges of the plates. I labeled one circle “DA” and the other “O”. 2 microliters of 0.5M sodium azide was added to the circle on both sides of the plate and 2 microliters of the blue diacetyl (DA) solution was added to the circle labeled “DA” (UCSB 2017). The sodium azide is used to kill the worms once they come into contact with it. Next, I obtained my RNAi plates and four filter screens. I added 750 microliters of water to each RNAi plate and gently swirled the water, then I removed the worms using a micropipette. I placed a Kimwipe under the filter screen and added the worms drop by drop. I then rinsed the worms by adding 200 microliters of DI water to the filter. I then added the worms to the chemotaxis plate by inverting the filter screen onto the corresponding plate and recorded the time of addition. I repeated this process for all four RNAi plates. After 60 minutes I counted the number of adult worms on both sides of the chemotaxis plate and recorded my results. Using these numbers, I calculated the chemotaxis index (CI) by dividing the number of worms on the “DA” side of the plate by the total number of worms on the plate. A chemotaxis index close to 1 means that chemotaxis in the worms still functioned. A low chemotaxis index indicates that the chemotaxis in the worms no longer functioned, or the genes knocked down in the worms were possibly involved in chemotaxis (UCSB 2017). ResultsThis section is a summary of the results from the experiment.Treatment Effects:Table 1. Chemotaxis Assay Results PlateNumber of Worms on “DA” side (A)Number of Worms on “O” side (B)Chemotaxis Index,P, (A)/(A+B)ODR-10 (positive control)2100.166L4440 (negative control)1070.588Experimental 1(ref-2)240.333Experimental 2(T26C5.2)000 Table 2. Summary of Class Chemotaxis indexes. This table summarizes the information obtained by all students in the MCDB 1A lab.RNAi CloneMean CISample SizeStandard DeviationODR-100.7233023548070.226248734L44400.7241983078270.213919956ref-20.716940426940.184095025T26C5.20.756213115610.216188777Statistical Analysis:Using the equation Z=(P1-P2)P1(1-P1)n1+P2(1-P2)n2we calculated the Z score for each of the experimental genes where P1and P2are the mean values for the chemotaxis index of the experimental and negative control, respectively and n1andn2are the sample sizes for the experimental and negative control, respectively. For ref-2, there was not a significant difference in the mean CI for ref-2 and the mean CI for L4440 (Z(94)=-0.148138, p=0.11134). For T26C5.2, there was not a significant difference in the mean CI for T26C5.2 and the mean CI for L4440 (Z(61)=0.560395, p=0.57548).Comparing Chemotaxis Indexes:Figure 1. Comparison of CI for Positive and Negative Controls, and the Two Experimentals, ref-2 (C47C12.3) and T26C5.2 in C. elegans. The error bars are one standard deviation above and below each average CI. DiscussionThis section contains the interpretation of the results listed above. The low CI for ODR-10 indicates that the chemotaxis of the worms was reduced in the worms with this gene knocked down. A CI of around 0.5 for L4440 means that chemotaxis in the worms was unaffected, which is expected for the negative control. The low CI for ref-2 indicated that the worms response was altered, so the ref-2 gene is possibly involved in chemotaxis because knocking it down changed the worm’s behavior. Finally, the CI for T26C5.2 is 0. This occurred because there was contamination in the agar plates while they were incubating and the worms died. Table 2 is a summary of the class results for the chemotaxis assay. The average CI for ODR-10, L4440, ref-2, and T26C5.2 are all around 0.7. This is not very robust data, and an error likely occurred. The error could have come from contamination in the plates resulting in the death of the worms, or students may have included worms that were not adults in their count to determine the chemotaxis index. Figure 1 shows that the average CI for ref-2 is around 0.7 and the average CI for T26C5.2 is around 0.75. The probabilities of observing the listed Z scores for each experimental indicate that for ref-2, assuming that the gene has no effect on chemotaxis, the calculated Z score would be obtained in 11% of studies due to random error. For T26C5.2, assuming that the gene has no effect on chemotaxis would result in the Z score being obtained in 56% of studies due to random error. These results do not give a clear reason to reject the null hypothesis that the experimental genes would not have an effect on chemotaxis because the possibilities of obtaining the same results randomly are high. This means that there is not a clear reason to conclude that ref-2 and T26C5.2 are genes involved in chemotaxis. An unexpected finding in this study is the similarity of the average CI for the positive and negative controls. These are not very robust chemotaxis indexes, and we would predict that the negative control would be close to 1 and the positive control would be close to 0.5, however, the data resulted in an average CI of about 0.72 for both the positive and negative controls. This indicates that there was an error in the data used in this study. A few possibilities of error are that the plates were contaminated or that students did not only count adult worms while doing their chemotaxis assays. These skewed CIs for the controls mean that when we compare our results to the controls, the information that we obtain is not completely correct because our average CIs were not robust. Therefore, the differences in our experimentals between the positive and negative controls is about the same, and deciding whether or not our hypothesis is correct is unclear. This leads to the inability to conclude whether the experimental genes were involved in chemotaxis.Research shows that ODR-10 is a gene known to be involved in chemotaxis, and using RNAi to knock down the gene results in reduced chemotaxis function (Sengupta et al. 1996). Our positive control was the ODR-10 gene, but the chemotaxis index did not indicate that the expression of the gene had been knocked down. This is an error in our data that does not allow us to make an accurate comparison between our positive control and our experimental genes. Therefore there is not a clear reason to conclude that ref-2 and T26C5.2 are genes involved in chemotaxis in C. elegans. This experiment was performed on C. elegans because they are a model organism, but the results can be analyzed to understand molecular genetics and systems biology (Gray and Cutter 2014). The entire genomic sequence of C. elegans is known, and the function of many genes has been uncovered through the use of RNAi (Fraser et al. 2000). The data obtained in this lab is contributing to the assignment of function to several genes in C. elegans. The worms are distantly related to humans, but all animals have a common origin that share development patterns and make studying the worms useful in learning about fields impacting human biology (UCSB 2017). Some protein families in both C. elegans and humans show close relationships (Grishok et al. 2001). This experiment was important because any information gathered about the functions of the experimental genes can be applied to biology in a larger sense than just in chemotaxis in C. elegans. ConclusionThe data that I obtained in this experiment leads us to believe that ref-2 is possibly involved in chemotaxis, but the average of the class data indicates that ref-2 is possibly uninvolved in chemotaxis. The worms in my experiment for T26C5.2 died, but the class data indicates that the gene is possibly not involved in chemotaxis. These assumptions are not confident as the chemotaxis indexes for the positive and negative controls were essentially the same, therefore not permitting a confident conclusion about the effects of knocking down ref-2 and T26C5.2.