BU06:Proposal

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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 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. 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. 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. RESEARCH I. Background The identification of naturally occurring bioluminescence systems in marine bacteria have 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. 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 expression. 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 stress. 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. 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. This involves the isolation of the lux operon as well as modification in order to abide by the standardized model for idempotent biological parts. 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 XbaI and EcoRI sites have been identified within the coding region of the luxCDABE operon as sequenced by Ed Meighan (Genbank M90093). 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. In abiding by 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. Likewise, 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. 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. 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. Following the standard assembly of biobricks, 5 parts from the registry will be combined in order to replicate the light sensing inhibitor. BBa_I15009, BBa_I15008 are necessary for the biosynthesis of PCB which binds to the ompR. 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. 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 AND 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 a yearly stable structure for the group as well as yearly funds to ensure that the group always has base funds 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. We are approaching two types of companies: those companies who work directly in synthetic biology and are committed to advancing this particular field and larger local companies in the area interested in funding innovative projects as part of their larger educational and community grants. The team is looking into the feasibility of selling tee-shirts to team members, parents, students, and faculty to raise funds. These would also serve the dual purpose of providing and 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 a enlightening interdisciplinary project that bring together students from all specializations in Engineering.

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