Boston University

From 2007.igem.org

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== About Us ==
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[[Image:BU_banner.JPG|center]]
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Welcome to the wiki for Boston University's iGEM 2007 team!
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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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== About Us ==
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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 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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Welcome to the wiki for Boston University's iGEM 2007 team!
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[[Image:BU_bubio.jpg|right]]
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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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== Our Project Plan ==
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Our project is aimed at increasing current production from [http://www.shewanella.org/ Shewanella oneidensis] by directed evolution of global transcription factors.
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Our plan so far:
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# Mutate global transcription factors
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## Design primers for amplification
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## Perform error-prone PCR
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# Transform mutated genes into E. coli
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## Choose plasmids
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## Restriction enzyme digestion
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## Ligation
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## Transformation into E. coli
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# Conjugate E. coli with Shewanella
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# Screen/select Shewanella strains for increased current production due to mutations.  Potential methods:
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## Alginate (?) beads and fluorocytometer
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## Metallo-Antibiotics
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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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== Week's (Ambitious) Goals ==
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Wednesday 5/30
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# Get all protocols
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#  Identify materials/prepare order
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#  Design Primers
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#  Learn about budget/POs
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Thursday 5/31
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# Do primer order
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# Start conjugation practice
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# Confirm restriction enzymes, ligases
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# Order confirmed/needed materials
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# Team Revew Meeting
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# Draft Thank-You Letters for our Sponsors
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Friday 6/1
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# Evaluate/continue conjugation, practice electroporation for E. coli
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# Revise proposal to include possibility of screening with alginate beads and fluorocytometer
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# Meeting with Tim:  Budgets/protocols, Pfizer/fundraising, iGEM registration, beads
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== What We've Accomplished ==
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Well we're basically finished.  I mean, cloning's been done a billion times, so this should be a piece of cake...I think.
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== Materials We Need ==
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Primers:  Need to Buy
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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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Error-Prone PCR:  Need to Buy
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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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Plasmids:  Need to Buy?
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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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Restriction Enzymes:  Need to Buy?
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== Our Project==
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Ligases:  Need to Buy?
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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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== Short-Term To-Do List ==
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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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Lab Orientation: Completed
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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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*[[Lab Orientation Checklist]]
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Lab Safety Training: Not Completed (Dave, Rahul, and Christian are scheduled for a training session on 5/30/07.  Danny will be taking it next week)
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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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Design of Primers:  Not Completed (Dave, please send me info about the finished primers when ready)
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electrogenesis even further.
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Ordering of Primers:  Not Completed
 
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Gathering of Protocols:  Not Completed (Chris, please send me the protocols when they are gathered)
 
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Ordering of Error-Prone PCR Materials: Not completed
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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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Thank-You Letters to iGEM Funders:  Not Completed
 
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== Question and Answer ==
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[[Image:fluorescent1.jpg]]
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[[Image:fluorescent2.jpg]]
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[[Image:fluorescent4.jpg]]
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[[Image:facs.jpg]]
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[[Image:topo.jpg]]
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[[Image:topo2.jpg]]
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[[Image:bulogo.jpg]]
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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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== Relevant Publications and Links==
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==Supported By==
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http://www.shewybase.bu.edu
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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"