Boston University

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== About Us ==
== About Us ==
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Welcome to the wiki for Boston University's iGEM 2007 team!
Welcome to the wiki for Boston University's iGEM 2007 team!
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[[Image:BU_bubio.jpg|right]]
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Our team consists of David Shi, Rahul Ahuja, Christian Ling, and Danny Bellin, all soon-to-be juniors majoring in Biomedical Engineering at Boston University.
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Our team consists of [[Boston_University/DS | David Shi]], [[Boston_University/RA | Rahul Ahuja]], [[Boston_University/CL | Christian Ling]], and [[Boston_University/DB | Danny Bellin]],
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all soon-to-be juniors majoring in Biomedical Engineering at Boston University.
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We are advised by [http://www.bu.edu/dbin/bme/faculty/?prof=tgardner Dr. Timothy Gardner], Assistant Professor of Biomedical Engineering, as well as Frank Juhn, Kevin Litcofsky, and Stephen Schneider, students in the [http://gardnerlab.bu.edu/ Gardner Laboratory], where we work.  We are grateful to our advisors for their time and support!
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We are advised by [http://www.bu.edu/dbin/bme/faculty/?prof=tgardner Dr. Timothy Gardner], Assistant Professor of Biomedical Engineering, as well as [[Boston_University/FJ | Frank Juhn]], [[Boston_University/KL | Kevin Litcofsky]], and [[Boston_University/SS | Stephen Schneider]], students in the [http://gardnerlab.bu.edu/ Gardner Laboratory], where we work.  We are grateful to our advisors for their time and support!
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We are also grateful to [http://www.pfizer.com Pfizer], the [http://www.bu.edu/eng Boston University College of Engineering], and the [http://www.bu.edu/eng/bme Boston University Deaprtment of Biomedical Engineering], for their generous support of our team.
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We are also grateful to [http://www.pfizer.com Pfizer], the [http://www.bu.edu/eng Boston University College of Engineering], and the [http://www.bu.edu/eng/bme Boston University Department of Biomedical Engineering], for their generous support of our team.
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== Our Project Plan ==
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== Our Project==
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Electrogenic microbes are microscopic organisms capable of extracting electrons from organic or chemical material and passing these electrons to extracellular organic or inorganic substrates.  The continued extraction and transfer of these electrons results, by definition, in usable electric current.  While electrogenic capacities have been observed among multiple bacteria species, Shewanella oneidensis is of particular interest due to its ability to oxidize virtually any carbon source. Research is currently underway into exploiting S. oneidensis for microbial fuel cells driven by ubiquitous material like waste and cellulose.
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The goal of our project is to use [[Directed_Evolution | directed evolution]] to increase the current output of the electrogenic bacteria [[Shewanella_oneidensis | Shewanella oneidensis]] (affectionately referred to as Shewie in the Gardner Lab). As the name suggests, directed evolution consists of two main steps: intentionally mutating DNA and then selecting for the expression of desired traits.  
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[[Image:Fig3.jpg|thumb|A microbial fuel cell]]
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In the case of S. oneidensis, certain [[GTRs_in_S_oneidensis | global transcription regulators]] in its genome have been identified as being related to the metabolic processes of the bacteria. These global transcription regulators will be mutated via [[Error_prone_PCR | error-prone PCR]] and transformed into S. oneidensis in hopes of altering current output.
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S. oneidensis-based fuel cells will be improved if the microbe’s own electrogenic capability is improved through manipulation of genes controlling current production.  The main problem, however, is determining how genetic modifications will affect an organism, as the interactions among genes in a single organism form a highly complex circuit network that is not readily resolved.  To address this problem, Dr. Timothy Gardner and his lab at Boston University have developed an algorithm called CLR to map the transcriptional regulatory interactions in microbial genomes.  Once CLR produces a map, effects of genetic engineering will be much more predictable, and those modifications leading to the most beneficial result will be easier to identify.  The following is an example of a transcriptional network inferred by CLR for just one of S. oneidensis’s metabolic processes.
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Bacteria that express greater electrogenic capability will then be selected via [[Boston_University/Fluorescence-Activated_Cell_Sorting | flow cytometry]] or other viable selection methods.  
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[[Image:Fig1.jpg|thumb|An example of a transcriptional network inferred by CLR for just one of S. oneidensis’s metabolic processes]]
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This process of directed evolution can be repeated with previously selected S. oneidensis in order to increase the level of
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The Gardner Laboratory has used CLR to map 800 regulatory interactions controlling metabolism and electron transport in S. oneidensis.  Results from the work have revealed that the transcription regulators SO1415, ttrR, and hlyU affect the largest number of genes involved in these processes:
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electrogenesis even further.
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[[Image:Fig2.jpg|thumb|Ranking of global transcription regulators in S. oneidensis]]
 
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Because of their global regulatory role, we hypothesize that mutations of these three genes present a high chance of affecting S. oneidensis in significant ways and potentially enhancing electrogenic output.
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{| align="center" style="color:white;" border="1"
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|-
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| bgcolor="#990000" color="white" height="30pt" align="center" | '''Our Team'''
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| bgcolor="#990000" color="white" align="center" | '''Project Design'''
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| bgcolor="#990000" color="white" align="center" | '''Project Results'''
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| bgcolor="#990000" color="white" align="center" | '''Miscellany'''
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|- style="color:#990000;"
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| align="left" width="375pt" |
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:'''Undergraduate Students'''
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::[[Boston_University/RA | Rahul Ahuja]] • [[Boston_University/DB | Daniel Bellin]]
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::[[Boston_University/CL | Christian Ling]] • [[Boston_University/DS | David Shi]]
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:'''Graduate Advisors'''
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::[[Boston_University/Frank Juhn | Frank Juhn]] • [[Boston_University/Kevin Litcofsky | Kevin Litcofsky]]
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::[[Boston_University/Stephen Schneider | Stephen Schneider]]
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:'''Principal Advisor'''
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::[http://www.bu.edu/dbin/bme/faculty/?prof=tgardner Dr. Timothy Gardner]
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| align="left" width="350pt"|
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:'''[[Boston_University/Why_Shewie | Why ''S. oneidensis''?]] '''
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:'''Directed Evolution'''
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::1. [[Boston_University/Plasmid Selection and Design | Plasmid Selection and Design]]
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::2. [[Boston_University/Mutation of GTFs | Mutation of GTFs]]
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::3. Transformation of GTFs into Shewie
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::::[[Boston_University/TOPO Cloning | TOPO Cloning]] • [[Boston_University/Conjugation | Conjugation]] [[Boston_University/Zymo | Zymo]]
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::4. Selection Methods
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::::[[Boston_University/Microencapsulation | Microencapsulation ]]
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::::[[Boston_University/Redox-sensitive fluorescent dye | Redox-sensitive fluorescent dye]]
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::::[[Boston_University/Fluorescence-Activated Cell Sorting | Fluorescence-Activated Cell Sorting]]
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| align="left" width="250pt" |
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:'''[[Boston_University/Zymo Transformation Results | Zymo Transformation]]'''
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:'''[[Boston_University/Electroporation Results | Electroporation]]'''
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:'''[[Boston_University/Bacterial Conjugation Results | Bacterial Conjugation]]'''
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:'''[[Boston_University/Plasmid Customization | Plasmid Customization]]'''
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:'''[[Boston_University/TOPO Cloning Results | TOPO Cloning]]'''
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:'''[[Boston_University/TOPO Transformation Results | TOPO Transformation]]'''
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:'''[[Hi-Scores and Other Greatness]]'''
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| align="left" width="250" |
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:'''[[Boston_University/Project Progress | Project Progress]]'''
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:'''[[Boston_University/Protocols | Protocols]]
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:'''[[Boston_University/Lab Photos | Lab Photos]]
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<!--:'''[[Boston_University/Team Photos | Team Photos]]-->
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:'''[[Boston_University/BU Photos | BU Photos]]
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:'''[[Boston_University/Image Dump | Image Dump (56k stay away!)]]
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|-
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|}
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Our plan so far:
 
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# Transform mutated global transcription factors
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<!--
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## Choose plasmids
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[[Image:shewie.jpg]]
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## Choose restriction enzymes
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[[Image:shewie2.jpg]]
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## Design primers for amplification
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[[Image:pjq200.jpg]]
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## Perform error-prone PCR
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[[Image:conjugation.jpg]]
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# Transform mutated genes into E. coli
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[[Image:fluorescent1.jpg]]
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## Restriction enzyme digestion
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[[Image:fluorescent2.jpg]]
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## Ligation
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[[Image:fluorescent3.jpg]]
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## Transformation into E. coli
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[[Image:fluorescent4.jpg]]
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# Conjugate E. coli with Shewanella
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[[Image:facs.jpg]]
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# Screen/select Shewanella strains for increased current production due to mutations. Potential methods:
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[[Image:topo.jpg]]
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## Alginate (?) beads and fluorocytometer
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[[Image:topo2.jpg]]
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## Metallo-Antibiotics
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[[Image:bulogo.jpg]]
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## (DB's random idea): Could we take advantage of spectrophotometry?  Perhaps we could split our collection of mutants into different groups, measure their absorbances with spectrophotometry, and assume that the sample with the lowest absorbance contains mutants producing more electricity and therefore growing slower. We could then split this sample into different groups and repeat.  While there might be some inefficient strains in the successful broth samples, on the whole, the broth might be a good one for use in a fuel cell.  Problem: Low absorbance could be due to mutants losing viability. Potential Solution: Let initial sample grow for a while so all mutants unable to grow will die off, all mutants able to grow will thrive, and then perform the screen.
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[[Image:gardnerlogo.jpg]]
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[[Image:bu_collegeofengineering.jpg]]
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[[Image:gardner_logo.jpg]]
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[[Image:bubio.jpg]]
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!-->
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== Current Status ==
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==Supported By==
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[[Boston_UniversityStatus | Current Status]]
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{| align="center" style="color:white;"
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|-
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| width="150pt" | [[Image:BU_pfizer.gif]]
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| width="325pt" | [[Image:bu_collegeofengineering.gif]]
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|[[Image:Bu_Gardner_logo_small.jpg]]
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|-
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|}

Latest revision as of 22:29, 20 October 2007

BU banner.JPG


About Us

Welcome to the wiki for Boston University's iGEM 2007 team!

BU bubio.jpg

Our team consists of David Shi, Rahul Ahuja, Christian Ling, and Danny Bellin, all soon-to-be juniors majoring in Biomedical Engineering at Boston University.

We are advised by [http://www.bu.edu/dbin/bme/faculty/?prof=tgardner Dr. Timothy Gardner], Assistant Professor of Biomedical Engineering, as well as Frank Juhn, Kevin Litcofsky, and Stephen Schneider, students in the [http://gardnerlab.bu.edu/ Gardner Laboratory], where we work. We are grateful to our advisors for their time and support!

We are also grateful to [http://www.pfizer.com Pfizer], the [http://www.bu.edu/eng Boston University College of Engineering], and the [http://www.bu.edu/eng/bme Boston University Department of Biomedical Engineering], for their generous support of our team.

Our Project

The goal of our project is to use directed evolution to increase the current output of the electrogenic bacteria Shewanella oneidensis (affectionately referred to as Shewie in the Gardner Lab). As the name suggests, directed evolution consists of two main steps: intentionally mutating DNA and then selecting for the expression of desired traits.

In the case of S. oneidensis, certain global transcription regulators in its genome have been identified as being related to the metabolic processes of the bacteria. These global transcription regulators will be mutated via error-prone PCR and transformed into S. oneidensis in hopes of altering current output.

Bacteria that express greater electrogenic capability will then be selected via flow cytometry or other viable selection methods.

This process of directed evolution can be repeated with previously selected S. oneidensis in order to increase the level of

electrogenesis even further.


Our Team Project Design Project Results Miscellany
Undergraduate Students
Rahul Ahuja • Daniel Bellin
Christian Ling • David Shi
Graduate Advisors
Frank Juhn • Kevin Litcofsky
Stephen Schneider
Principal Advisor
[http://www.bu.edu/dbin/bme/faculty/?prof=tgardner Dr. Timothy Gardner]
Why S. oneidensis?
Directed Evolution
1. Plasmid Selection and Design
2. Mutation of GTFs
3. Transformation of GTFs into Shewie
TOPO Cloning • Conjugation Zymo
4. Selection Methods
Microencapsulation
Redox-sensitive fluorescent dye
Fluorescence-Activated Cell Sorting
Zymo Transformation
Electroporation
Bacterial Conjugation
Plasmid Customization
TOPO Cloning
TOPO Transformation
Hi-Scores and Other Greatness
Project Progress
Protocols
Lab Photos
BU Photos
Image Dump (56k stay away!)


Supported By

BU pfizer.gif Bu collegeofengineering.gif Bu Gardner logo small.jpg
Retrieved from "http://2007.igem.org/wiki/index.php/Boston_University"