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Introduction to Biomedicine for Engineers

by: Louie Nikolaus

Introduction to Biomedicine for Engineers BIO ENG 10

Marketplace > University of California - Berkeley > Bioengineering > BIO ENG 10 > Introduction to Biomedicine for Engineers
Louie Nikolaus

GPA 3.53

I. Conboy

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I. Conboy
Class Notes
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This 4 page Class Notes was uploaded by Louie Nikolaus on Thursday October 22, 2015. The Class Notes belongs to BIO ENG 10 at University of California - Berkeley taught by I. Conboy in Fall. Since its upload, it has received 29 views. For similar materials see /class/226551/bio-eng-10-university-of-california-berkeley in Bioengineering at University of California - Berkeley.


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Date Created: 10/22/15
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 oflacI 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 cAMPCAT complex which controls the rate ofprodution increases tremendously B To prevent expression oflacZ 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 oflacZ 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 lad 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 lad 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 N3oxohexanoylLhomoserine 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 ofGFP Nguyen Vivian SID 21123610 A B This synthetic circuit uses a CreLox system where the transcription factor is activated by UV light and binds to the Cre promoter driving the transcription of Cre Once the Cre recombinase protein is produced it binds to the manufactured Lox sites cleaving out the quotSTOPquot sequence ofDNA and allowing enhanced yellow uorescent protein or EYFP to be produced Because Cre recombinase deleted the quotSTOPquot sequence in the DNA EYFP will continue being produced even after the UV light is gone Nguyen Vivian SID 21123610 4 A Iwould select the RCE1 and ZMFSTE24 enzymes for molecular evolution in order to generate a more functional while still mutant lamin A RCE1 and ZMFSTE24 control the C terminal cleavage but for people with HutchinsonGilford Progeria Syndrome they lack the recognition site for RCE1 and ZMFSTE24 to bind to and cleave off By mutating RCE1 and ZMFSTE24 to recognize farnesylprelamin A even without the recognition site normally located within the 50 amino acid deletion they could cleave it and turn it into functional lamin A B To generate a library of enzymes using random mutagenesis primers can be changed one nucleotide at a time to form point mutations These primers will still be complementary enough to attach to the DNA but will alter the sequence of amino acids altering the RCE1 and ZMFSTE24 enzymes Each enzyme in the library will have a different point mutation C D To translate this to a therapy create multitudes of the mutant enzyme for injection into a HutchinsonGilford Cterminals and creating functional lamin A But there are many precautions that must come with this treatment Without further testing it is not known exactly where the mutant RCE1 and ZMFSTE24 enzymes will attach to the DNA Without that knowledge vital proteins maybe not be expressed due to the enzymes covering that part of the DNA halting the transcription of that gene


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