BU06:Executive Summary

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[[Category: Boston University 2006| Boston University 2006]]
[[Category: Boston University 2006| Boston University 2006]]
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''' ''Proposal Teams: Please copy your material into the appropriate section here by Monday night June 19th, so we can all be ready for a group edit on Tuesday.'' '''
+
'''Proposal outline'''
 +
*Intro
 +
**Igem and synth. biology
 +
**previous successful projects and media reviews
 +
**who we are and what are our goals
 +
**prove igem concept
 +
**contribute novel parts to catalog (registry)
 +
**produce a working project
 +
*Project specifics
 +
**the plan: simple project, expected good results, extensions
 +
**technical: what it is: see Melissa's
 +
**results: modeling, characterizing, documenting in data sheet
 +
*materials/ lab req.
 +
**costs and time line
 +
*Fundraising cost, goals & sources
 +
**Goals and sources
 +
*timeline
 +
**lab training, lab planning - max people in shifts at lab -> lab teacher
 +
**schedule
-
'''I. Intro'''
 
-
The International Genetically Engineered Machines competition (iGEM) is built around the central question of whether biological systems can be built from standard interchangeable parts and operated in living cells. iGEM was developed by Massachusetts Institute of Technology professors, primarily Tom Knight and Drew Endy, with their students, and is still hosted at MIT. Over the past three years of structured synthetic biology competitions, iGEM, has developed a "Registry" of hundreds of available and working parts, called "BioBricks". These parts are designed to be inserted into microorganisms to achieve human goals. The ultimate goal is to guide this area of molecular biology from a difficult and complicated series of individual experiments to a standardized engineering discipline, approachable to any engineer by using an online registry, similar to the McMaster-Carr catalog of electromechanical components.
+
''' ''Proposal Writing Teams: Please copy your material into the appropriate section below by Monday night June 19th, so we can all be ready for a group edit on Tuesday.'' '''
-
In past years, teams have made such contributions as a bacterial "photographic film," a caffeine detector, and an inverter analogous to an electronic inverter. The competition has grown rapidly from five teams in the initial year, to 13 teams, to 40 international teams in this year's field of competitors. iGEM has been earning attention in the scientific and general press, as much for the success of its projects as for its unique pedagogical goals of bringing together undergraduates and graduates from widely divergent scientific and engineering backgrounds. Past coverage has included Nature, Science, and this year has been featured as the cover story of a recent issue of Scientific American.
 
-
BU's 2006 iGEM team is composed of a group of primarily undergraduate biomedical engineering students, reinforced with graduate students in a number of disciplines, including biology, electrical engineering and physics. Most have completed an introductory course in molecular biology and have some lab experience. Our Principal Investigator is Dr. Timothy Gardner, whose early work in the field of synthetic biology resulted in the development of the first biological toggle switch.
+
Boston University iGEM 2006 Proposal
 +
Billy Andre, Alec Cerchiari, Mackenzie Cowell, Nancy Mendonca Davis, Kevin Duong,
 +
Alex Peterson, Camilo Garcia, Nadav Ivzan, Frank Juhn, Alex Kates, Melissa Kinney,
 +
Felix Liu, Avi Oza Umer Rathore, Christine Woods
-
Our goals in participating in the competition are to help prove the basic iGEM principles by producing a functional biologically engineered system, contribute a novel bioluminescent part to the Registry, develop our molecular design skills, gain more experience in molecular biological laboratory techniques, and strengthen the synthetic biology community among schools and for the future growth of the industry. In addition, iGEM has been beneficial in providing thesis and research material for participants, and we hope to be able to take advantage of this to help guide our future academic careers.
 
-
We are convinced that our team has a plan designed for success. As a first-year team, we aspire to establish ourselves at the annual iGEM "Jamboree" as a major contender in the competition, and begin a tradition of Boston University iGEM excellence. The Jamboree is the primary opportunity for teams to present and promote their projects with documentation, results, and characterization of the biological systems they have built. We believe our participation will reflect well on ourselves and enhance BU's image as a front-runner in undergraduate synthetic biology. The support, academic, technical and financial, of the BU community will be a major component of our success.
 
-
'''II. Project Specifics'''
+
ABSTRACT
 +
Synthetic Biology is a field aimed at innovating and enhancing molecular bioengineering by standardizing molecular cloning methodologies. This effort to create modular designs in biology ultimately lends to greater efficiency and speed at which research may be conducted, while decoupling the biological and engineering design aspects. The International Genetically Engineered Machines competition (iGEM) engages undergraduate students, placing them at the forefront of synthetic biology and providing invaluable research experience. The Boston University iGEM 2006 team aims to contribute to this promising field by offering designs consisting of previously confirmed genetic parts as well as newly created parts. While the Registry of Standard Biological Parts is constantly evolving, the Boston University team seeks to make an important contribution to the current catalog of genetic reporters. The luxCDABE operon provides bioluminescence without the need for stimulus or growth in special media. In the form of a BioBrick, the large operon becomes more accessible, as it is no more difficult to ligate into an existing plasmid than other single-gene reporters. This construct will be proven useful in the context of modifying past research in order to create a novel light controlled bioluminescent system. The team, under the advising of Principal Investigator Dr. Timothy Gardner and the support of graduate students, seeks to begin a tradition of excellence for Boston University at the annual iGEM jamboree in November. As a new student-led organization within the BU community, we are seeking financial, academic and technical support from BU faculty and administration as well as corporate sponsorship.
-
'''III. Materials/Lab Requirements'''
 
-
'''IV. Fundraising Costs, Goals & Rources'''
+
INTRODUCTION
 +
The International Genetically Engineered Machines competition (iGEM) is built around the central question of whether biological systems can be built from standard interchangeable parts and operated in living cells. iGEM was developed by Massachusetts Institute of Technology professors, primarily Tom Knight and Drew Endy, with their students, and is still hosted at MIT. Over the past three years of structured synthetic biology competitions, iGEM, has contributed to a "Registry of Standard Biological Parts," which contains hundreds of available and working genetic parts, called "BioBricks". These parts are designed with the intent of controlling cellular function in microorganisms to achieve human-defined goals. The ultimate aim is to guide this area of molecular biology from a difficult and complicated series of individual experiments to a standardized engineering discipline, approachable to any engineer by using an open-source online registry, similar to the McMaster-Carr catalog of industrial components.
-
'''V. Timeline'''
+
In past years, teams have made such contributions as a bacterial "photographic film," a caffeine detector, and an inverter analogous to an electronic inverter. The competition has grown rapidly from five teams in the initial year, to 13 teams, to 40 international teams in this year's field of competitors. iGEM has been earning attention in the scientific and general press, as much for the success of its projects as for its unique pedagogical goals of bringing together undergraduates and graduates from widely divergent scientific and engineering backgrounds. Past coverage has included Nature, Science, and this year iGEM has been featured as the cover story of a recent issue of Scientific American.  
 +
BU's 2006 iGEM team is composed primarily of a group of undergraduate biomedical engineering students, reinforced with graduate students in a number of disciplines, including biology, electrical engineering and physics. Most have completed an introductory course in molecular biology and have some lab experience. Our Principal Investigator is Dr. Timothy Gardner, whose work in the field of synthetic biology has resulted in the development of the first artificial biological toggle switch.
 +
Our team goals in participating in the competition are to help prove the basic iGEM principles by producing a functional biologically engineered system, contribute a novel bioluminescent part to the Registry, develop our molecular design skills, gain more experience in molecular biological laboratory techniques, and enhance the cooperation of the synthetic biology community among schools and for the future growth of the industry. In addition, iGEM experience has been beneficial in providing thesis and research material for participants, and we hope to be able to take advantage of this to help guide our future academic careers.
 +
 +
We are convinced that our team has a plan designed for success. As a first-year team, we aspire to establish ourselves at the annual iGEM "Jamboree" as a major contender in the competition, and begin a tradition of Boston University iGEM excellence. The Jamboree is the primary opportunity for teams to present and promote their projects with documentation, results, and characterization of the biological systems they have built. We believe our participation will reflect well on ourselves and enhance BU's image as a front-runner in undergraduate synthetic biology. The support, academic, technical and financial, of the BU community will be a major component of our success.
 +
 +
 +
RESEARCH
 +
 +
I. Background
 +
 +
The identification of naturally occurring bioluminescence systems in marine bacteria has led to a class of genetic reporters with a variety of applications. Reporters are commonly used in transformation and transfection assays, promoter characterization, and gene expression analysis. In the lux system, bioluminescence occurs through a chemical reaction in which a luciferin is oxidized by a luciferase. This reaction allows nondestructive analysis of gene expression because the light emission generates little thermal radiation. While the single genes encoding for green fluorescent protein and beta-galactosidase have been widely used as a reporters, the lux operon provides noninvasive bioluminescence as it does not require stimulus or growth in special medium. Moreover, the lux system can be grown in typical aerobic conditions because it requires only the native components of O2, ATP, reduced flavin mononucleotide, and NADPH for expression1. The luxCDABE operon has been identified as a method of expressing bioluminescence under the control of a promoter of interest. Therefore, potential applications of drug and chemical detection have led to systems monitoring antibiotic efficacy in vivo, groundwater contamination, the presence of mercury, and oxidative stress2,3,4.
 +
 +
The potential applications of the lux bioluminescence system as well as the complexity of the operon highlight the need for standardization. Establishing the lux operon as a BioBrick would be an important contribution to the current list of reporters in the Registry. The length of the luxCDABE operon, which spans almost 7kb, presents problems for ordinary lab techniques. As a BioBrick, the entire operon could be added through a simple established procedure. This increases the ease of combining the operon with promoters or other genes of interest. Similarly, this standardization alters the level of abstraction in order to shift the focus from molecular details to a systems engineering design approach. Through the clearly defined functions of BioBricks, the genetic engineering process becomes decoupled so that in-depth understanding of individual genes and functions is not necessary; parts engineering requires molecular background, while engineering design works to create devices by combining BioBricks, which can be thoroughly characterized in data sheets to enable optimal usage. This system allows people of many different backgrounds to work together in creating cells with desired functions. The contribution of a BioBrick containing the luxCDABE operon presents a standardized method which can be applied to both future and past projects in order to create new systems.
 +
 +
II. Goals
 +
 +
The Boston University iGEM team seeks to contribute this novel luxCDABE BioBrick to the Registry of Standard Biological Parts, and build a functioning "Biological Night-Light" system with it to enter into the iGEM competition. This involves the isolation of the lux operon as well as modification in order to abide by the standardized model for idempotent biological parts established for all BioBricks in the Registry. The BioBrick design is constructed of a coding region flanked on either side by two distinct restriction sites in order to homogenize the process of ligating parts into an existing plasmid. The properties of restriction sites XbaI and SpeI allow site specific ligation while also creating an irreversible mixed site. The lux BioBrick will be used in conjunction with existing parts and devices in order to create a new functional system. The light-sensing construct created by registry parts will be assembled using the lux operon as a reporter, creating aphotic responsive luminescence.
 +
 +
III. Methods
 +
 +
Silent Point Mutation of Restriction Site
 +
XbaI and EcoRI sites have been identified within the coding region of the luxCDABE operon (figure 1) as sequenced by Ed Meighan (Genbank M90093)5. EcoRI cuts at the ends of the sequence (+2 and -4). XbaI cuts in the middle of the luxD gene (+2411). In order to remove the XbaI restriction site, a silent point mutation will be introduced using site directed mutagenesis. The sequence which codes for leucine within the XbaI site will be changed from CTA to CTU, maintaining the identity of the amino acid. The Stratagene QuikChange XL kit utilizes the PfuTurbo DNA polymerase in order to extend oligonucleotide primers containing the mutation, resulting in efficient site directed mutagenesis. The luxCDABE operon will be isolated from the pUC19 plasmid through restriction digest with EcoRI, thus eliminating the sites on either end; the resulting sticky ends will not form new sites unless ligated with other EcoRI sites.
 +
 +
BioBrick Standardization
 +
To achieve the standard format for the BioBrick construct, regions containing two specific restriction sites must be added to each end of the operon. The length of the operon, 6960 base pairs, creates problems in adding these regions. Typical methods of insertion include designing PCR primers which include the desired addition. However, standard DNA polymerases are only effective to around 5000bp. Therefore, two possible methods have been identified. It is possible to perform multiple PCR reactions on different regions of the operon which overlap at a central restriction site. The restriction site AgeI has been identified at 3388bp. These products can then be digested and ligated in order to obtain the complete operon region. Another possible approach involves synthesis of the desired insertion and subsequent sticky or blunt end ligation. The restriction site for MfeI has an overlapping region which is complementary to that of EcoRI. However, when ligated, this would create a mixed site, unrecognized by either enzyme.
 +
 +
BioBrick Testing and Characterization
 +
The successful formation of the luxCDABE BioBrick construct allows ligation into a plasmid containing the kanamycin and/or ampicillin resistance genes. In order to test the viability, a constitutive promoter (BBa_I14032) as well as a ribosomal binding site (BBa_B0030) will be ligated upstream of luxCDABE. Likewise, a terminator (BBa_B0015) will be included downstream (figure 2). This results in constant expression of bioluminescence when transformed in E. coli. This system can be characterized based upon several variables such as growth rate and temperature.
 +
 +
 +
"Biological Night-Light" System
 +
Following the standard assembly of BioBricks (figure 3), 5 parts from the registry will be combined in order to replicate the light sensing inhibitor developed for a former iGEM entry.6 BBa_I15009 and BBa_I15008 are necessary for the biosynthesis of PCB which binds to ompR, a cytoplasmic DNA-binding protein. The cph1 light receptor, BBa_I15010, inhibits the creation of Envz, which in turn phosphorylates ompR. The overall reporter gene is controlled by the promoter from BBa_R0082, which depends on ompR. When phosphorylated ompR binds, it activates transcription. In the original construct, the reporter gene is lacZ (BBa_I2012). Therefore, lacZ is expressed when there is no light; in the presence of light EnvZ is repressed, ompR is not phosphylated, and therefore transcription does not occur. This system can also be characterized to determine the effect of different light and other optimal conditions. In the context of our system, the lacZ gene can be replaced by luxCDABE in order to create bacteria which emit bioluminescence in the dark. The overall device will be analyzed with respect to the bioluminescence under the control of the constitutive promoter.
 +
 +
IV. Timeline
 +
 +
Due to the size of the group, 4 teams have been established, each consisting of one Team Leader, who has experience with molecular cloning and mutagenesis protocols. Lab training sessions will be conducted by these leaders, with only 3 people in the lab at a time. The lab training will consist of all procedures necessary in order to perform the experiments, including miniprep, digestion, ligation, PCR, electrophoresis, transformation, clean up of PCR and gel products, and other general lab techniques. Tentatively, training is scheduled to begin in early to mid July, as materials arrive. Immediately after training, groups will begin work on small individual research goals, which make up the larger experiment. Lab work will continue through the end of October, in preparation for the annual Jamboree in November.
 +
 +
 +
 +
FUNDRAISING COSTS, GOALS, & SOURCES
 +
 +
The BU iGEM team has ambitious fundraising goals but is confident we will attain our goals with our thorough fundraising plan. We have obtained starting funds that ensure our research will not be held back while we raise all the funds needed to complete the entire project.
 +
 +
The BU iGEM team has begun the process of applying for student organization status from the Student Activities Office which will provide an ongoing stable structure for the group as well as yearly funding to ensure that the group always has a financial basis from which to operate. The group will be notified of acceptance and funds in early October.
 +
 +
The team is pursuing educational grants from several companies interested in funding innovative and collaborative projects such as iGEM. Two types of companies have been identified as prospects: those companies which work directly in synthetic biology and are committed to advancing this particular field and larger companies in the Boston area interested in funding innovative projects as part of their corporate educational and community grants.
 +
 +
The team is considering some small-scale fundraising on campus, such as selling promotional items to raise awareness as well as funds. This would also serve the dual purpose of providing an impressive presence at the November Jamboree.
 +
 +
The iGEM team is also hopeful that administrators, professors, and labs in the Engineering Department at Boston University will provide funds to the team in recognition of the great publicity that our successful participation in this competition will bring to the BU Engineering Department and for the opportunity this program brings to the students in our department. iGEM provides beneficial hands-on research experience to undergraduates in preparation for their Senior Project and also exposes students to one of the most cutting-edge fields in Biomedical Engineering. It is also an enlightening interdisciplinary project that bring together students from all specializations in Engineering.
 +
 +
REFERENCES
 +
 +
 +
1. Van Dyk, T. K., B. L. Ayers, R. W. Morgan, and R. A. LaRossa. 1998. Constricted flux through the branched-chain amino acid biosynthetic enzyme acetolactate synthase triggers elevated expression of genes regulated by rpoS and internal acidification. J. Bacteriol. 180:785-792.
 +
 +
2. Francis, K. P., J. Yu, C. Bellinger-Kawahara, D. Joh, M. J. Hawkinson, G. Xiao, T. F. Purchio, M. G. Caparon, M. Lipsitch, and P. R. Contag. 2001. Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon. Infect. Immun. 69:3350-3358.
 +
 +
3. Applegate, B. M., S. R. Kehrmeyer, and G. S. Sayler. 1998. A chromosomally based tod-luxCDABE whole-cell reporter for benzene, toluene, ethylbenzene, and xylene (BTEX) sensing. Appl. Environ. Microbiol. 64:2730-2735
 +
 +
4. Van Dyk TK, Majarian WR, Konstantinov KB, Young RM, Dhurjati PS, LaRossa RA. 1994. Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions. Appl Environ Microbiol. 60 (5): 1414-1420.
 +
 +
5. Meighen,E.A. and Szittner,R.B. 1992. Multiple repetitive elements and organization of the lux operons of luminescent terrestrial bacteria. J. Bacteriol. 174 (16): 5371-5381.
 +
 +
6. Anselm Levskaya, Aaron A. Chevalier, Jeffrey J. Tabor, Zachary Booth Simpson, Laura A. Lavery, Matthew Levy, Eric A. Davidson, Alexander Scouras, Andrew D. Ellington, Edward M. Marcotte and Christopher A. Voigt. 2005. Synthetic biology: Engineering Escherichia coli to see light. Nature 438: 441-442.
 +
 +
 +
MATERIALS
 +
Chemicals:
 +
Vendor Catalog Number Total
 +
LB Media EMD 1.10285.0500 $35.77
 +
LB Agar EMD 1.10283.0500 $78.40
 +
Antibiotics Fisher BP1760-5 $46.45
 +
Antibiotics Fisher BP906-5 $47.17
 +
*Ethanol Fisher S739852 $14.95
 +
*TE Fisher AC32734-5000 $49.70
 +
PCR master mix Stratagene 600640 $329.00
 +
Restriction enzymes/buffers NEB $300.00
 +
Agarose American Bioanalytical AB00972 $36.40
 +
TAE Fisher BP1332-500 $49.96
 +
1Kb ladder Fisher BP2579-100 $117.71
 +
T4 Ligase/buffer NEB M0202S $63.00
 +
*Glucose Fisher S76789 $13.10
 +
Glycerol Fisher BP229-1 $69.83
 +
Primers IDT $90.00
 +
Molec. Bio H20 Fisher 07-200-574 $60.00
 +
 +
Kits:
 +
Miniprep kit QIAGEN 27104 $70.00
 +
PCR purification kit QIAGEN 28104 $84.00
 +
Gel extraction kit QIAGEN 28704 $84.00
 +
Mutagenesis Kit Stratagene 200517 $206.00
 +
 +
Disposables:
 +
**Petri dishes Fisher MS-D13-00325 $32.00
 +
**Gloves Fisher S473384 $49.75
 +
Pipet tips Fisher 21-277-2B $169.20
 +
Pipet tips Fisher 02-707-100 $155.88
 +
Pipet tips Fisher 21-197-8A $178.65
 +
Pipet tips Fisher 07-200-574 $94.29
 +
*Microcentrifuge tubes Fisher 05-406-18 $30.80
 +
Microcentrifuge tubes Fisher 21-236-66 $60.11
 +
Round bottom tubes Fisher 14-959-11B $173.10
 +
Cryogenic Vial Fisher 10-500-26 $38.63
 +
Parafilm Fisher S37441 $15.40
 +
Equipment:
 +
Pipetters
 +
Bunsen burner
 +
Centrifuge
 +
PCR machine
 +
Gel apparatus
 +
Voltage supply
 +
Water bath
 +
Glassware
 +
Tube racks
 +
Wire loop
 +
Glass rod
 +
37ºC incubator
 +
37ºC shaker
 +
Gel imaging system
 +
4ºC Fridge
 +
-20ºC freezer
 +
-80ºC freezer
 +
Autoclave
 +
Cold room
----
----
Line 122: Line 265:
**the plan: simple project, expected good results, extensions
**the plan: simple project, expected good results, extensions
**technical: what it is: see Melissa's
**technical: what it is: see Melissa's
-
**results: modeling, characterizing
+
**results: modeling, characterizing, documenting in data sheet
*materials/ lab req.
*materials/ lab req.
**costs and time line
**costs and time line
Line 129: Line 272:
*timeline  
*timeline  
**lab training, lab planning - max people in shifts at lab -> lab teacher
**lab training, lab planning - max people in shifts at lab -> lab teacher
-
**scheduale
+
**schedule

Latest revision as of 00:00, 25 June 2006


Proposal outline

  • Intro
    • Igem and synth. biology
    • previous successful projects and media reviews
    • who we are and what are our goals
    • prove igem concept
    • contribute novel parts to catalog (registry)
    • produce a working project
  • Project specifics
    • the plan: simple project, expected good results, extensions
    • technical: what it is: see Melissa's
    • results: modeling, characterizing, documenting in data sheet
  • materials/ lab req.
    • costs and time line
  • Fundraising cost, goals & sources
    • Goals and sources
  • timeline
    • lab training, lab planning - max people in shifts at lab -> lab teacher
    • schedule


Proposal Writing Teams: Please copy your material into the appropriate section below by Monday night June 19th, so we can all be ready for a group edit on Tuesday.


Boston University iGEM 2006 Proposal Billy Andre, Alec Cerchiari, Mackenzie Cowell, Nancy Mendonca Davis, Kevin Duong, Alex Peterson, Camilo Garcia, Nadav Ivzan, Frank Juhn, Alex Kates, Melissa Kinney, Felix Liu, Avi Oza Umer Rathore, Christine Woods


ABSTRACT

Synthetic Biology is a field aimed at innovating and enhancing molecular bioengineering by standardizing molecular cloning methodologies. This effort to create modular designs in biology ultimately lends to greater efficiency and speed at which research may be conducted, while decoupling the biological and engineering design aspects. The International Genetically Engineered Machines competition (iGEM) engages undergraduate students, placing them at the forefront of synthetic biology and providing invaluable research experience. The Boston University iGEM 2006 team aims to contribute to this promising field by offering designs consisting of previously confirmed genetic parts as well as newly created parts. While the Registry of Standard Biological Parts is constantly evolving, the Boston University team seeks to make an important contribution to the current catalog of genetic reporters. The luxCDABE operon provides bioluminescence without the need for stimulus or growth in special media. In the form of a BioBrick, the large operon becomes more accessible, as it is no more difficult to ligate into an existing plasmid than other single-gene reporters. This construct will be proven useful in the context of modifying past research in order to create a novel light controlled bioluminescent system. The team, under the advising of Principal Investigator Dr. Timothy Gardner and the support of graduate students, seeks to begin a tradition of excellence for Boston University at the annual iGEM jamboree in November. As a new student-led organization within the BU community, we are seeking financial, academic and technical support from BU faculty and administration as well as corporate sponsorship.


INTRODUCTION

The International Genetically Engineered Machines competition (iGEM) is built around the central question of whether biological systems can be built from standard interchangeable parts and operated in living cells. iGEM was developed by Massachusetts Institute of Technology professors, primarily Tom Knight and Drew Endy, with their students, and is still hosted at MIT. Over the past three years of structured synthetic biology competitions, iGEM, has contributed to a "Registry of Standard Biological Parts," which contains hundreds of available and working genetic parts, called "BioBricks". These parts are designed with the intent of controlling cellular function in microorganisms to achieve human-defined goals. The ultimate aim is to guide this area of molecular biology from a difficult and complicated series of individual experiments to a standardized engineering discipline, approachable to any engineer by using an open-source online registry, similar to the McMaster-Carr catalog of industrial components.

In past years, teams have made such contributions as a bacterial "photographic film," a caffeine detector, and an inverter analogous to an electronic inverter. The competition has grown rapidly from five teams in the initial year, to 13 teams, to 40 international teams in this year's field of competitors. iGEM has been earning attention in the scientific and general press, as much for the success of its projects as for its unique pedagogical goals of bringing together undergraduates and graduates from widely divergent scientific and engineering backgrounds. Past coverage has included Nature, Science, and this year iGEM has been featured as the cover story of a recent issue of Scientific American.

BU's 2006 iGEM team is composed primarily of a group of undergraduate biomedical engineering students, reinforced with graduate students in a number of disciplines, including biology, electrical engineering and physics. Most have completed an introductory course in molecular biology and have some lab experience. Our Principal Investigator is Dr. Timothy Gardner, whose work in the field of synthetic biology has resulted in the development of the first artificial biological toggle switch.

Our team goals in participating in the competition are to help prove the basic iGEM principles by producing a functional biologically engineered system, contribute a novel bioluminescent part to the Registry, develop our molecular design skills, gain more experience in molecular biological laboratory techniques, and enhance the cooperation of the synthetic biology community among schools and for the future growth of the industry. In addition, iGEM experience has been beneficial in providing thesis and research material for participants, and we hope to be able to take advantage of this to help guide our future academic careers.

We are convinced that our team has a plan designed for success. As a first-year team, we aspire to establish ourselves at the annual iGEM "Jamboree" as a major contender in the competition, and begin a tradition of Boston University iGEM excellence. The Jamboree is the primary opportunity for teams to present and promote their projects with documentation, results, and characterization of the biological systems they have built. We believe our participation will reflect well on ourselves and enhance BU's image as a front-runner in undergraduate synthetic biology. The support, academic, technical and financial, of the BU community will be a major component of our success.


RESEARCH

I. Background

The identification of naturally occurring bioluminescence systems in marine bacteria has led to a class of genetic reporters with a variety of applications. Reporters are commonly used in transformation and transfection assays, promoter characterization, and gene expression analysis. In the lux system, bioluminescence occurs through a chemical reaction in which a luciferin is oxidized by a luciferase. This reaction allows nondestructive analysis of gene expression because the light emission generates little thermal radiation. While the single genes encoding for green fluorescent protein and beta-galactosidase have been widely used as a reporters, the lux operon provides noninvasive bioluminescence as it does not require stimulus or growth in special medium. Moreover, the lux system can be grown in typical aerobic conditions because it requires only the native components of O2, ATP, reduced flavin mononucleotide, and NADPH for expression1. The luxCDABE operon has been identified as a method of expressing bioluminescence under the control of a promoter of interest. Therefore, potential applications of drug and chemical detection have led to systems monitoring antibiotic efficacy in vivo, groundwater contamination, the presence of mercury, and oxidative stress2,3,4.

The potential applications of the lux bioluminescence system as well as the complexity of the operon highlight the need for standardization. Establishing the lux operon as a BioBrick would be an important contribution to the current list of reporters in the Registry. The length of the luxCDABE operon, which spans almost 7kb, presents problems for ordinary lab techniques. As a BioBrick, the entire operon could be added through a simple established procedure. This increases the ease of combining the operon with promoters or other genes of interest. Similarly, this standardization alters the level of abstraction in order to shift the focus from molecular details to a systems engineering design approach. Through the clearly defined functions of BioBricks, the genetic engineering process becomes decoupled so that in-depth understanding of individual genes and functions is not necessary; parts engineering requires molecular background, while engineering design works to create devices by combining BioBricks, which can be thoroughly characterized in data sheets to enable optimal usage. This system allows people of many different backgrounds to work together in creating cells with desired functions. The contribution of a BioBrick containing the luxCDABE operon presents a standardized method which can be applied to both future and past projects in order to create new systems.

II. Goals

The Boston University iGEM team seeks to contribute this novel luxCDABE BioBrick to the Registry of Standard Biological Parts, and build a functioning "Biological Night-Light" system with it to enter into the iGEM competition. This involves the isolation of the lux operon as well as modification in order to abide by the standardized model for idempotent biological parts established for all BioBricks in the Registry. The BioBrick design is constructed of a coding region flanked on either side by two distinct restriction sites in order to homogenize the process of ligating parts into an existing plasmid. The properties of restriction sites XbaI and SpeI allow site specific ligation while also creating an irreversible mixed site. The lux BioBrick will be used in conjunction with existing parts and devices in order to create a new functional system. The light-sensing construct created by registry parts will be assembled using the lux operon as a reporter, creating aphotic responsive luminescence.

III. Methods

Silent Point Mutation of Restriction Site XbaI and EcoRI sites have been identified within the coding region of the luxCDABE operon (figure 1) as sequenced by Ed Meighan (Genbank M90093)5. EcoRI cuts at the ends of the sequence (+2 and -4). XbaI cuts in the middle of the luxD gene (+2411). In order to remove the XbaI restriction site, a silent point mutation will be introduced using site directed mutagenesis. The sequence which codes for leucine within the XbaI site will be changed from CTA to CTU, maintaining the identity of the amino acid. The Stratagene QuikChange XL kit utilizes the PfuTurbo DNA polymerase in order to extend oligonucleotide primers containing the mutation, resulting in efficient site directed mutagenesis. The luxCDABE operon will be isolated from the pUC19 plasmid through restriction digest with EcoRI, thus eliminating the sites on either end; the resulting sticky ends will not form new sites unless ligated with other EcoRI sites.

BioBrick Standardization To achieve the standard format for the BioBrick construct, regions containing two specific restriction sites must be added to each end of the operon. The length of the operon, 6960 base pairs, creates problems in adding these regions. Typical methods of insertion include designing PCR primers which include the desired addition. However, standard DNA polymerases are only effective to around 5000bp. Therefore, two possible methods have been identified. It is possible to perform multiple PCR reactions on different regions of the operon which overlap at a central restriction site. The restriction site AgeI has been identified at 3388bp. These products can then be digested and ligated in order to obtain the complete operon region. Another possible approach involves synthesis of the desired insertion and subsequent sticky or blunt end ligation. The restriction site for MfeI has an overlapping region which is complementary to that of EcoRI. However, when ligated, this would create a mixed site, unrecognized by either enzyme.

BioBrick Testing and Characterization The successful formation of the luxCDABE BioBrick construct allows ligation into a plasmid containing the kanamycin and/or ampicillin resistance genes. In order to test the viability, a constitutive promoter (BBa_I14032) as well as a ribosomal binding site (BBa_B0030) will be ligated upstream of luxCDABE. Likewise, a terminator (BBa_B0015) will be included downstream (figure 2). This results in constant expression of bioluminescence when transformed in E. coli. This system can be characterized based upon several variables such as growth rate and temperature.


"Biological Night-Light" System Following the standard assembly of BioBricks (figure 3), 5 parts from the registry will be combined in order to replicate the light sensing inhibitor developed for a former iGEM entry.6 BBa_I15009 and BBa_I15008 are necessary for the biosynthesis of PCB which binds to ompR, a cytoplasmic DNA-binding protein. The cph1 light receptor, BBa_I15010, inhibits the creation of Envz, which in turn phosphorylates ompR. The overall reporter gene is controlled by the promoter from BBa_R0082, which depends on ompR. When phosphorylated ompR binds, it activates transcription. In the original construct, the reporter gene is lacZ (BBa_I2012). Therefore, lacZ is expressed when there is no light; in the presence of light EnvZ is repressed, ompR is not phosphylated, and therefore transcription does not occur. This system can also be characterized to determine the effect of different light and other optimal conditions. In the context of our system, the lacZ gene can be replaced by luxCDABE in order to create bacteria which emit bioluminescence in the dark. The overall device will be analyzed with respect to the bioluminescence under the control of the constitutive promoter.

IV. Timeline

Due to the size of the group, 4 teams have been established, each consisting of one Team Leader, who has experience with molecular cloning and mutagenesis protocols. Lab training sessions will be conducted by these leaders, with only 3 people in the lab at a time. The lab training will consist of all procedures necessary in order to perform the experiments, including miniprep, digestion, ligation, PCR, electrophoresis, transformation, clean up of PCR and gel products, and other general lab techniques. Tentatively, training is scheduled to begin in early to mid July, as materials arrive. Immediately after training, groups will begin work on small individual research goals, which make up the larger experiment. Lab work will continue through the end of October, in preparation for the annual Jamboree in November.


FUNDRAISING COSTS, GOALS, & SOURCES

The BU iGEM team has ambitious fundraising goals but is confident we will attain our goals with our thorough fundraising plan. We have obtained starting funds that ensure our research will not be held back while we raise all the funds needed to complete the entire project.

The BU iGEM team has begun the process of applying for student organization status from the Student Activities Office which will provide an ongoing stable structure for the group as well as yearly funding to ensure that the group always has a financial basis from which to operate. The group will be notified of acceptance and funds in early October.

The team is pursuing educational grants from several companies interested in funding innovative and collaborative projects such as iGEM. Two types of companies have been identified as prospects: those companies which work directly in synthetic biology and are committed to advancing this particular field and larger companies in the Boston area interested in funding innovative projects as part of their corporate educational and community grants.

The team is considering some small-scale fundraising on campus, such as selling promotional items to raise awareness as well as funds. This would also serve the dual purpose of providing an impressive presence at the November Jamboree.

The iGEM team is also hopeful that administrators, professors, and labs in the Engineering Department at Boston University will provide funds to the team in recognition of the great publicity that our successful participation in this competition will bring to the BU Engineering Department and for the opportunity this program brings to the students in our department. iGEM provides beneficial hands-on research experience to undergraduates in preparation for their Senior Project and also exposes students to one of the most cutting-edge fields in Biomedical Engineering. It is also an enlightening interdisciplinary project that bring together students from all specializations in Engineering.

REFERENCES


1. Van Dyk, T. K., B. L. Ayers, R. W. Morgan, and R. A. LaRossa. 1998. Constricted flux through the branched-chain amino acid biosynthetic enzyme acetolactate synthase triggers elevated expression of genes regulated by rpoS and internal acidification. J. Bacteriol. 180:785-792.

2. Francis, K. P., J. Yu, C. Bellinger-Kawahara, D. Joh, M. J. Hawkinson, G. Xiao, T. F. Purchio, M. G. Caparon, M. Lipsitch, and P. R. Contag. 2001. Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon. Infect. Immun. 69:3350-3358.

3. Applegate, B. M., S. R. Kehrmeyer, and G. S. Sayler. 1998. A chromosomally based tod-luxCDABE whole-cell reporter for benzene, toluene, ethylbenzene, and xylene (BTEX) sensing. Appl. Environ. Microbiol. 64:2730-2735

4. Van Dyk TK, Majarian WR, Konstantinov KB, Young RM, Dhurjati PS, LaRossa RA. 1994. Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions. Appl Environ Microbiol. 60 (5): 1414-1420.

5. Meighen,E.A. and Szittner,R.B. 1992. Multiple repetitive elements and organization of the lux operons of luminescent terrestrial bacteria. J. Bacteriol. 174 (16): 5371-5381.

6. Anselm Levskaya, Aaron A. Chevalier, Jeffrey J. Tabor, Zachary Booth Simpson, Laura A. Lavery, Matthew Levy, Eric A. Davidson, Alexander Scouras, Andrew D. Ellington, Edward M. Marcotte and Christopher A. Voigt. 2005. Synthetic biology: Engineering Escherichia coli to see light. Nature 438: 441-442.


MATERIALS

Chemicals: Vendor Catalog Number Total LB Media EMD 1.10285.0500 $35.77 LB Agar EMD 1.10283.0500 $78.40 Antibiotics Fisher BP1760-5 $46.45 Antibiotics Fisher BP906-5 $47.17

  • Ethanol Fisher S739852 $14.95
  • TE Fisher AC32734-5000 $49.70

PCR master mix Stratagene 600640 $329.00 Restriction enzymes/buffers NEB $300.00 Agarose American Bioanalytical AB00972 $36.40 TAE Fisher BP1332-500 $49.96 1Kb ladder Fisher BP2579-100 $117.71 T4 Ligase/buffer NEB M0202S $63.00

  • Glucose Fisher S76789 $13.10

Glycerol Fisher BP229-1 $69.83 Primers IDT $90.00 Molec. Bio H20 Fisher 07-200-574 $60.00

Kits: Miniprep kit QIAGEN 27104 $70.00 PCR purification kit QIAGEN 28104 $84.00 Gel extraction kit QIAGEN 28704 $84.00 Mutagenesis Kit Stratagene 200517 $206.00

Disposables:

    • Petri dishes Fisher MS-D13-00325 $32.00
    • Gloves Fisher S473384 $49.75

Pipet tips Fisher 21-277-2B $169.20 Pipet tips Fisher 02-707-100 $155.88 Pipet tips Fisher 21-197-8A $178.65 Pipet tips Fisher 07-200-574 $94.29

  • Microcentrifuge tubes Fisher 05-406-18 $30.80

Microcentrifuge tubes Fisher 21-236-66 $60.11 Round bottom tubes Fisher 14-959-11B $173.10 Cryogenic Vial Fisher 10-500-26 $38.63 Parafilm Fisher S37441 $15.40 Equipment: Pipetters Bunsen burner Centrifuge PCR machine Gel apparatus Voltage supply Water bath Glassware Tube racks Wire loop Glass rod 37ºC incubator 37ºC shaker Gel imaging system 4ºC Fridge -20ºC freezer -80ºC freezer Autoclave Cold room

PUNCHY!

  • SB is a new field - Why is it interesting, no, essential!?
    • Standardization of genetic engineering to enable innovation
    • Improves collaboration, rate of innovation,
    • 'programmable' cells
    • when we do genetic engineering it should be like building a bridge--we can show that what we build will work; it's not a wild-ass guess whether or not it will work
    • specialization -- let people focus on their chosen area of expertise with the assumption that all the other levels of abstraction will just work
    • iGEM is about undergrads

Motivation factors for BU professors:

  • put boston university undergraduates at the center of an infrastructure of a 'revolutionary' (sorry) new field
  • opportunity to build a year-on-year franchise (a resource for getting kids molecular biology experience -- keep in mind there's not that many _mol bio_ labs in the dept)
  • let our undergrads socialize scientifically outside the university
  • complement/enhance senior project (one of our key differentiators--sorry to lapse into admin-speak)
  • bringing an engineering sensibility to biology is a huge deal for a biomedical engineering department centrally located in a city with a huge biomedical industrial complex
  • Jay Keasling/Amyris greatly reduced the cose of producing artemesin for huge # of people in need -- as the good Dr. Endy says we should make this routine so that it doesn't require $20m of Bill Gates' money and 100 (?) man-years of effort.
  • quantitative quantitative quantitative (BU BME professors' three favorite words)
  • growth in # of teams (5, one of which was BU) -> 12 -> 43 -> at least seven hundred next year
  • obvious opportunity for press coverage
  • biofactories
  • we have people doing tissue engineering; wouldn't they like to have predictable cells?
  • the Biological Century


We need one million dollars!

awesome proposal team!!! i posted the outline and additional points we came up with on the proposal meeting. when you write up the executive summary don't be intimidated to be as creative as possible. there is no true or false in this write up. more over feel comfortable to post/save your summaries on this page (with your name at the bottom so we will have a common place to go over them together in the meeting, cut and paste the appropriate parts and have a place to observe eachother work and comment outside of our get togethers. When we will meet on friday we will collect the ideas we like from every summary and put it all together into an awesome summary. (Nadav 06/13/06)

  • proposal
    • convincing that we are scientific/professional
    • more specifics of plan
    • success stories
    • actual literature
    • road map
    • celebrity backers
    • list of tools and materials
    • how much money we need
  • Brochure
    • what is synthetic biology
    • how does igem relate
    • potential excitement success
    • what we are doing?
  • why?
    • reasons to help- prestige and senior project
    • opportunities for undergraduates
  • how
    • road map
    • list of needs
    • tools, materials, practical ways to help

We are a group of Undergraduate students from BU who participated in a molecular biology course during the Spring 2006 semester. Our professor, Timothy Gardner, and Professor Drew Endy (MIT) introduced us to the world of synthetic biology. Synthetic Biology is a new field of research that integrates science and engineering in order design biological systems that carry out specific functions, such as programming bacteria to work as a biological toggle switch. Fascinated by the opportunities given by this novel we field of research we decided to participate in the iGEM 2006 competition. Because of the complexity involved in designing and building biological systems we decided to attempt to implement three ideas, each more complicated than the previous one. Moreover, the more complicated ideas are built off of the simpler ideas


Synthetic Biology is the new field of biological engineering aimed at innovating and enhancing all bioengineering by introducing standardizations of methods and results. This effort to create modular designs in biology ultimately lends to greater efficiency and speed at which research and engineering may be conducted. In specifying biological parts, engineers may be able to more accurately predict the outcomes of a design, as well as using and contributing to all other work in Synthetic Biology. The fundamentals of this field are grounded in the collaborative interaction between engineers and researchers. This is materialized in the annual iGEM competition at MIT. The Boston University iGEM 2006 team aims to contribute to this promising field by offering designs consisting of previously confirmed genetic parts as well as newly created parts.


The BU iGEM team, a group of undergraduate researchers from Boston University, are very excited by the opportunities presented by the third annual International Genetically Engineered Machines Competition (iGEM). iGEM is an international competition between Universities, in which groups of undergraduates from different schools work to create an innovative and useful biological system. The standardization of these bio-systems as parts, formally called biobricks, is at the forefront of synthetic biology. Biological engineers use these biobricks to develop new biological systems more efficiently. The possibilities are endless. The BU team hopes that through their work in iGEM, they can help in the effort to reduce the cost and increase the accessibility of synthetic biology technologies. As a starting point, the team has designed a bio-system that will produce light under specific conditions. The design requires the creation of a new biobrick for light emission, which is then combined with a light detection biobrick created and added to the registry of parts for iGEM 2005. In order to complete our design we will require financial backing. Thank you for your time and consideration.


the 21st century introduces synthetic biology! igem - international genetichaly engineered machine competition is an anual cosmopolitan competition participated by schools from all around the world that is designed to promote the novel and rapidaly expanding field of thynthetic biology with the goals of standarizing it and promoting free information sharing. the competition is going to be held for the fourth time in november in an event called "The Jamboree" that is held at MIT. we are a group of students from boston University who decided to form agroup and participate in the competition backed by our world known faculty and professors, Jim Collins and Timothy Gardner we came up with exciting and novel ideas we would like to develop for the competition starting with a biological night light, biological chemical detector and light excited bacteria. developments from previous igem competitions include biological photophilm and biological counter. all of our ideas are based on the chemotaxis protein and luxR operon and require the help of synthetic biology companies to synthesis the sequences needed. in order to develop our ideas we need funding of $10,000 in order to buy the raw materials and essential lab equipment in return to the funding we offer publication and fame for the companies who are willing to support us, more over a spectacular insight to the world of synthetic biology as a whole and igem as the core of it.

Nadav (06/15/06)

proposal outline

  • Intro
    • Igem and synth. biology

-previous successful projects and media reviews

    • who we are and what are our goals

-prove igem concept -contribute novel parts to catalog (registry) - produce a working project

  • Project specifics
    • the plan: simple project, expected good results, extensions
    • technical: what it is: see Melissa's
    • results: modeling, characterizing, documenting in data sheet
  • materials/ lab req.
    • costs and time line
  • Fundraising cost, goals & sources
    • Goals and sources
  • timeline
    • lab training, lab planning - max people in shifts at lab -> lab teacher
    • schedule
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