Download Gene Control: lac Operon and Synthetic Circuits in Genetic Engineering - Prof. I. M. Conbo and more Study notes Biology in PDF only on Docsity! Nguyen, Vivian SID #: 21123610 BioE 10: Problem Set 1 1. A) To express both lacZ and lacY genes at high levels of glucose and low levels of lactose, artificially manufactured small interfering RNA, or siRNAs can be inserted into a cell by liposomes. These siRNAs will be coded to have the same sequence as the gene for lacI. Once inside the cytoplasm, a RNA Induced Silencing Complex, or RISC, binds to the siRNA therefore silencing it and any sequence complementary to it. This will silence the expression of lacI at the translational step. Without the expression of lacI, no repressor proteins will be created, and expression of lacZ and lacY commence. After silencing lacI, adenylyl cyclase must be mutated so that it remains active independent of the glucose levels. This can be achieved by inserting tRNA with mutated amino acids that will change the sequence of amino acids that compile the adenylyl cyclase enzyme. With adenylyl cyclase producing cAMP from ATP all the time, the cAMP-CAT complex, which controls the rate of prodution, increases tremendously. B) To prevent expression of lacZ and lacY genes at low levels of glucose and high levels of lactose, the repressor protein encoded by lacI should be mutated so that lactose is unable to bind to it. The repressor protein could be mutated through the tRNA method also, by introducing synthetic tRNA with mutated amino acids that will change the sequence of the amino acids of the repressor protein. Without the ability to attach to lactose, the repressor protein will always remain attached preventing expression of lacZ and lacY under all conditions. C) To express GFP under the control of the Lac promoter only in low glucose and high lactose, insert the DNA gene for GFP downstream of the lacI and promoter region, for example between the promoter and the lacZ gene, or the lacZ gene and the lacY gene. This must be done in vitro and then inserted into an E. coli cell via plasmid vector or liposome. Once inside, restriction enzymes and ligase can also be inserted to ensure that our synthetic DNA is incorporated into the genome. D) To express GFP under the control of the Lac promoter only in low glucose and low lactose, insert the DNA gene for GFP downstream of the lacI and promoter region in vitro and insert into E. coli, just as for question 1C. Once this plasmid is incorporated into the genome, insert siRNAs encoded for the repressor protein into the cell by liposomes. This will cause RISC to silence the expression of the repressor protein leading to the production of GFP along with lacZ and lacY. Nguyen, Vivian SID #: 21123610 2. A) The signal is the autoinducer N-3-oxohexanoyl-L-homoserine, also known as AHL. B) The biosensor module is the LuxR protein. C) The engineered gene regulatory network is: D) The output is the expression of GFP.
