7.014 Quiz III Handout • Quiz III: Friday, April 22 12:05 - 12:55 Walker Gym **This will be a closed book exam** • Quiz Review Session: Wednesday, April 20 7:00 - 9:00 pm room 54-100 • Open Tutoring Session: Thursday, April 21 4:00 - 6:00 pm room 66-1562 Question 1 Shown below is the portion of the diagram from page 9 that shows the electron transport pathway. In this pathway, electrons are transferred from NADH to O2; the energy of this reaction is coupled to pumping of H+. NADH + H+(FADH2)NAD+(FAD)2 e-O2H2O2 e- H+ (in space A) H+ (in space B)electron transportproton pumping2 e-drug Xdrug Y2 e- Under normal circumstances, cells carrying out respiration: • consume O2 • produce CO2 • produce 36 ATP per glucose consumed Two drugs, drug X and drug Y, can pick up electrons from particular intermediates in this pathway. This is shown above. a) You treat cells carrying out respiration with a saturating dose of drug X, so that all the electrons which would normally continue along the pathway are captured by drug X. Under these conditions: • Will the cells continue to consume O2? • Will the cells continue to produce CO2? • Will the rate of ATP synthesis increase or decrease or stay the same? Explain your reasoning. b) You treat cells carrying out respiration with a saturating dose of drug Y, so that all the electrons which would normally continue along the pathway are captured by drug Y. Under these conditions: • Will the cells continue to consume O2? • Will the cells continue to produce CO2? • Will the rate of ATP synthesis increase or decrease or stay the same? Explain your reasoning.3 Question 2 Agrobacterium tumefaciens is a bacterium that infects a wide range of broad-leaved plants. Most of the Agrobacterium genes are found on its large chromosome, but some of the genes that allow this bacterium to infect plant cells are found on the bacterial Ti plasmid. During infection, the bacterium transfers the Ti plasmid DNA to the plant and the plasmid DNA is integrated into the genome of the plant. This new DNA encodes plant hormones that stimulate cell division of the infected cells. This excessive cell division produces a tumor. The Ti plasmid also carries the genes responsible for opine production. Opines are modified amino acids that only plant cells infected with Agrobacteria produce. These opines can be used by Agrobacteria as the SOLE carbon and energy source, and are not used by other organisms. a) Circle the correct answer… Agrobacterium is an autotroph or a heterotroph. b) For the Ti plasmid DNA to have an effect within the plant cell, an appropriate promoter must be included for each gene. i) What is a promoter? ii) Describe the potential problem(s) of trying to express genes from a eukaryote in a prokaryote. iii) Would the genes carried on the Agrobacterium Ti plasmid have a prokaryotic or eukaryotic promoter? Why? iv) What is the likely origin of the Ti plasmid DNA sequences? The major staple food for hundreds of millions of people is rice. However, rice lacks carotenoids that are converted into beta-carotene, a precursor of vitamin A. Millions of people suffer from vitamin A deficiencies. Rice has most of the biochemical pathway for producing beta-carotene, but is lacking two enzymes. Using Agrobacterium tumefaciens and modified Ti plasmids, scientists have created rice plants that have the needed enzymes and can make beta-carotene. These plants produce yellowish colored rice grains, called Golden Rice, which is high in beta-carotene.4 Question 2, continued To create Golden Rice plants, two different Ti plasmids were made. In the first case, the Ti plasmid carried the crt1 gene from the bacterium E. uredovora. The other Ti plasmid carried the daffodil gene psy2. You want to recreate the first of these modified Ti plasmids. c) You are given E. uredovora genomic DNA and want to use this to make a library in E. coli cells. You purchase the following cloning vector for use in constructing your library. It encodes the gene for ampicillin resistance (ampR). PvuII: 5'-CAG|CTG-3' EcoRI cuts: 5'-G|AATTC-3' 3'-GTC|GAC-5' 3'-CTTAA|G-5' i) With what enzyme will you cut your cloning vector? ii) With what enzyme will you cut your genomic DNA? d) You successfully cut both your genomic DNA and your cloning vector. You mix them together and add DNA ligase. i) In what other cellular process have we encountered DNA ligase? ii) What does DNA ligase do? e) You transform E. coli cells with your ligated DNA to make a library. i) The E. coli cells that you use for transformation should have what phenotype prior to being transformed? ii) Once transformed, how do you isolate the E. coli cells that have a plasmid away from the E. coli cells that don’t have a plasmid? f) You successfully create a wild type genomic DNA library from E. uredovora. Assume that production of beta-carotene is essential for growth of E. uredovora cells on minimal media. How could you use your library to clone the crt1 gene by complementation? EcoRI EcoRI Cloning PvuII ori amp R 3000 bp5 Question 3 Take the following simple ecosystem enclosed in a fish tank: ••••••••phytoplanktonherbivorousfishwatergravelheterotrophicbacteria(one species)•theonly energy in pu t is ligh t• the fish eat only the phytoplankton• humans harvest the fish• nothing eats the bacteria• the whole tank, including the sedim en ts, is aerobic a) Construct an energy flow model for this ecosystem. i) Label all compartments and products ii) assign a different label to each of the energy flows iii) describe what each energy flow represents. b) Suppose you added a worm to this ecosystem that only consumes waste produced by the fish. What would be the effect on the level of respiration by the heterotrophic bacteria (increase, decrease, stay the same) of adding this worm to the tank, if the worm's waste and detritus was not consumed by the detritivores. Explain your reasoning briefly. c) Suppose you added a worm to this ecosystem that only consumes waste produced by the fish. What would be the effect on bacterial respiration (increase, decrease, stay the same) of adding this worm to the tank, if the worm's waste was consumed by the
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