Lab Report

Pathogens can spread widely and affect many organisms at the same time. Several organisms evolve to become immune or to resist pathogens including humans and C. elegans. In this experiment, C. elegans avoidance assay plates were analyzed to determine if the C. elegans evolved to resist the pathogen S. marcerens.

RNA polymerase can bind to the promoter region of the DNA even when the lac repressor is bound to the operator site.

Similarly, the recipient E. Coli bacteria is resistant to nalidixic acid, and would be able to grow on a Nal plate, but not on the Cm plate. By plating the recipient bacteria on chloramphenicol, we can ensure that the sample was purely recipient if there is no growth.

Now that the Plasmid ‘psiRNA’ is transfected into the somatic cell, scientists don’t need to inject dsRNA into the cell each time to stop the production of BLG protein, the plasmid automatically produces siRNA into the cytoplasm

Plasmids are small double stranded circular non chromosomal DNA molecules containing their own origin of replication. Hence, they are capable of replication independent of the chromosomal DNA in bacteria. Plasmids present in one or more copies per cell, can carry extra chromosomal DNA from one cell to another cell and serve as tools to clone and manipulate genes. Plasmids used exclusively for this purpose are known as vectors. The genes of interest can be inserted into these vector plasmids creating a recombinant plasmid. Recombinant plasmids can play a significant role in gene therapy, DNA vaccination, and drug delivery [Rapley, 2000].

It is of upmost important that immune defenses work in a regular coordinated manner so that the host can fight with infection and a regulated immune system prevent the deleterious effects of unchecked immune responses on host cells [63]. As Caenorhabditis elegans is susceptible to infection by a variety of fungal and bacterial pathogens so it employs a highly coordinated innate immune system to detect and counter pathogen attack, no matter the attacking pathogen is ingested or comes into external contact with the animal [64, 65].

Elegans mutations are found to be well maintained within the ISS Insulin/IGF-1 pathway that can have a lead on a longer lifespan extension and this can help with age related diseases along with the improvement of health in later life, This is very important in the understanding of these age related neurodegenerative diseases because it will help with understanding of how people/patients could then go on to being helped with these diseases as well as along with tumour formations patients, These functions are looked at closely by the FoxO transcription factor known as DAF-16 using microarrays, bioinformatics predictions as well as SAGE ( serial analysis of gene expression) and DAF-16 is located downstream from the ISS Kinase cascade. Looking at how the DAF-16 target gene along with testing its functions has been a long process of over 10 years in the ageing

The plates in this photo were mislabeled. The plate labeled LB+ amp+ Arab (+) is actually the culture grown on LB+ amp+ IPTG(+). Furthermore, the plate labeled LB+ amp+ IPTG(+) is the culture grown on LB+ amp+ Arab (+). In figure II the plate reveals that the phenotype was displayed on the medium that contained IPTG, but it is labeled with Arab. This picture was taken with gel imaging system to visualize the phenotype. The plate that labeled Arab appears darker because this plate’s cells were displaying fluorescent light. Furthermore, this reveals that the transformed plasmid contained a promoter that was induced by IPTG. A hypothesis was made at this point of the experiment; the hypothesis was that pGlo was inserted into the cells, but this hypothesis was wrong. At this point of the experiment the labels were believed to be correct, but if the knowledge of the switched labels were know, the hypothesis would have been: pFG was inserted into the E. coli culture. The way the reporter gene was determined was through the physical analysis.

Clustered regulatory interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) are an immune response evolved by bacteria and archea as an adaptive defense mechanism to invading DNA. (4) The CRISPR Cas9 system relies on the uptake of invading DNA fragments that are then inserted into CRISPR loci. (4) In the CRISPR loci, repeats are separated by nucleotide spacers which match and or composed of invading DNA.(4) New spacer DNA is incorporated by Cas1 and Cas2.(4) The CRISPR spacer loci then transcribe into short CRISPR RNAs (crRNA) which anneal to foreign nucleic acids in conjunction with complementary binding trans-activating cr RNA(tracrRNA) to form a duplex which is then cleaved to provide a guiding RNA cr/tracr RNA hybrid.(4) the RNA hybrid acts as a guiding mechanism for Cas9 by complementary binding to the invading nucleotides.(4) Cas9 is an endonuclease that can cause a double stranded cleave in DNA(4) Cas9 guided with sgRNA then cleaves the foreign DNA resulting in double stranded breaks effectively disrupting and thereby removing a gene.(1)(2)(3)(4) After a ds break occurs cellular machinery attempts to fix the break with non homologous end joining in which cellular systems effectively sutures the broken ends of the DNA by recombining the remaining ends of DNA to once again produce a continuous strand.(4) This

It is triggered by Dicer, an enzyme that assists in the activation of the RNA induced silencing complex, an important component for RNAi in fungi, plants, and animals, according to the article, “Pathway Central: RNAi Pathway”. The process is first activated by dsRNA molecules and requires a specific set of gene products. The dsRNA is then cut into smaller pieces by the Dicer enzyme in an ATP-dependent reaction. The dsRNA in fungi, animals, and plants are exogenous, meaning that the RNA is directly sent to the cytoplasm and cut into shorter lengths by Dicer. As of yet, endogenously expressed dsRNA molecules have not been discovered in mammals, where the RNA would first be shaped into the stem- loop structure in the nucleus, then sent to the cytoplasm to be further modified by Dicer. In RISC, the enzyme Dicer also supplies the initial RNA material to activate the complex as well as the first RNA substrate molecule. RISC with a bound siRNA targets complementary mRNA molecules and degrades them, resulting in lower levels of protein translation and the eventual inhibition of the

1a. To select sRNA, mRNA, and lncRNA unique to Cn but not present in mammalian hosts.

The mechanism for RNAi follows one of two major pathways: one originating with foreign injected double-stranded RNA (dsRNA) and the other originating with micro-RNA

RNAi challenges this notion by demonstrating that RNA can also disrupt this flow of genetic information and prevent protein expression. Since 1998 the exact mechanism of RNAi has been thoroughly investigated, and it has been shown that the RNAi machinery, known as RNA-induced silencing complex (RISC), is activated when two fragments of RNA combine to form double-stranded RNA (Ramachandran et al., 2013). RISC then binds to one of these RNA fragments, which acts as a template allowing RISC to search for its complementary sequence on the target mRNA. As such, this fragment is known as the guide or ‘antisense’ strand (Wang et al., 2011). When RISC detects its matching sequence, the target mRNA is cleaved by certain nucleases or degraded by the cellular machinery, which effectively prevents translation of the transcript and silences expression of the protein (Wang et al., 2011). As described, the initial step in this process relies on the formation of an RNA duplex, which can occur in two pathways. Firstly, dsRNA can form in an endogenous manner due to the presence of non-coding regulatory RNA called micro (mi)RNA. miRNAs were discovered in the early 2000s as a result of Fire and Mello’s work, and they function as important post-transcriptional

The lux R, which has its own promoter and is transcribed in the opposite direction from the lux operon, could not be transcribed from the same strand because the RNA polymerase recognizes a promoter sequence only in the direction of 5’ to 3’, and the lux R gets transcribed in the opposite direction from the lux operon. Thus, the transcriptions of luxR and lux operon have to occur on two different strands including coding strand and template strand.

Caenorhabditis elegans have two loci encoding the chaperone Grp170, Grp170a and Grp170b. During ER stress, these two Grp loci are expressed differently in C. elegans (Rockwell 2015). The expression of grp170 mRNA was analyzed in nematodes deficient for either loci; the results