1. Your PhD project\'s aim is to discover a gene which can degrade \"Bisphenol A
ID: 182241 • Letter: 1
Question
1. Your PhD project's aim is to discover a gene which can degrade "Bisphenol A" (BA) which is a plastic related substance that is well known to be associated with a variety of diseases. If you discover this gene, you will express it in E. coli, purify it, and use t to decontaminate soil and water that is polluted by BA. You hypothesize that a bacteria that lives somewhere on earth might contain such a gene. To find this bacteria, you obtain soil (earth) samples from 5 different "garbage dumps": 1. Mamak, Ankara (MA); 2. Girne, Cyprus (GC) 3. Bodrum, Mugla (BM); 4 Umraniye, Istanbul (UI); 5. Harmandali, Izmir (HI). You then measure BA levels in the soil samples collected from each garbage dump. Your results are the following (values represent BA as g / 10 gram soil): MA: 1 I, GC: 10, BM: 9, UI: 0.001. HI: 8. a. Based on these results, which garbage dump might contain bacteria that can metabolize BA? Explain your logic very briefly b. You therefore, obtain soil from this garbage dump and grow them in the laboratory. But you don't know how many species of bacteria exist in this soil sample. Your boss tells you that you can analyze the coxl gene (ORF 600bp) to find out the number of species in this sample. He tells you that this is possible because all sequencing analysis performed so far for every bacterial species, show that nucleotides 1-50, 250-300 and 450-550 are conserved (identical) in all species. How can this information be useful in determining the number of bacterial species in this earth sample? Please describe the experiments you would perform to discover the number of bacterial species in this sample very briefly You have determined that there are only 4 bacterial species in this soil sample. Describe very briefly how you would clone each bacterial species separately c.Explanation / Answer
a. If a bacteria present in these dumps metabolize BA then eventually the levels of BA would be low. UI has the lowest levels of BA i.e. 0.001, therefore it will have the bacteria metabolizing BA.
b. It is given that the nucleotide 1-50, 250-300, 450-550 are conserved DNA sequences of cox1 gene and must be same in all bacterial species in the sample. Any two bacteria differing rest of the nucleotide position will represnt a different bacterial species i.e. 51-249, 301-449, 551-600. We can decipher this by simply sequencing the gene from different bacteria in the sample. This can be done by sanger's method using ddNTPs. Plate the bacteria from the samples on a suitable solid medium. If there is a visible difference among the colonies then, analyze them first make replicas and then select and isolate colonies and then go back to inncoulating them for isolating cox1 gene and deciphering the different bacterial species. This will tell you the number of differnt species, trce back to the colony you used to isolate the gene.
c. From the previous step you have on a solid medium the differnt bacterial species from the sample, pick a colony from each species, innoculate the liquid medium and the incubate. Once liquid media has becom turbid, you can plate it back to the solid media to clone the bacterial species.
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