Mr. J is trying tо design primers tо identify а fungus he isоlаted from а soil sample, through PCR followed by Sanger sequencing of a novel gene believed to be universally presented in fungi. He collated multiple sequences of this gene from the NCBI database that cover a broad range of fungal species. He found that three regions of ~25-120 base pairs seem identical (conserved) across the fungi, and four regions of 50-700bp that are highly variable: 1. Which two regions shall Mr. J target for primer design (i.e. regions where the forward and reverse primers anneal to) in order to maximize the sequence information for identifying the fungus? Please provide a rationale for your choice (4 points). 2. Before setting up PCR, Mr. J has to isolate total DNA from the fungus. Propose a DNA extraction method and a method to check DNA quality and quantity (2 points). Based on the answer from question 1, Mr. J designed primer pairs and ran the PCR. He tested different PCR parameters and examined the PCR products by gel electrophoresis: 3. Describe the issues indicated by the different scenarios (1-4) and explain a possible cause for each scenario. (4 points) 4. After optimizing the PCR, Mr. J obtains a PCR product of the correct size. Based on gene annotation this gene is likely protein-coding. The gene also has the appropriate restriction sites that can be cloned into a plasmid. He is using the pGEM T easy vector (below) and E. coli to produce the protein from the gene for further investigation. He decided to clone the gene into the middle of the LacZ gene. 5. Describe the key steps of the cloning experiments (6 points) and the roles of Ampr and LacZ genes in specific steps. (2 points) 6. After successfully cloning the protein-coding gene into the pGEM-T Easy vector and expressing it in E. coli, Mr. J observes unusual phenotypes in the E. coli, such as enhanced biofilm formation, indicating a potential metabolic shift. To investigate these metabolic changes, he decides to conduct a metabolomic analysis using High-Performance Liquid Chromatography (HPLC) coupled with Mass Spectrometry (MS). What are the key steps involved in conducting this metabolomic experiment? (6 points).