Team:Carnegie Mellon

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Contents 1 Introduction: Motivation and Background 2 Primary Objective: A New Set of Well-Characterized Promoters 3 Secondary Objective: Humanistic Practice 4 The Team 5 Further Considerations

Introduction: Motivation and Background

• Our primary goal is to develop new promoters that can be measured with fluorescent technology.
• We seek to develop a system that will allow researchers in the field of synthetic biology to accurately measure a variety of metrics in gene expression networks including translational efficiency and transcriptional strength.

• We hypothesize that we can use Spinach (a fluorescent RNA sequence) and a FAP (fluorogen activating protein) as biosensors to reflect these metrics in vivo (in living cells), rather than in vitro (in a test tube), which can be very costly and impractical.

• We will characterize the relationship between the rates of production of Spinach and FAP and the gene's translational efficiency and transcription rate.
  

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Project Description

Experimental

The design and implementation of synthetic biological systems often require information on transcription and translation rates and on the impact of both RNA and protein levels on metabolic activities of host cells. Such information is needed when both strong and low levels of expression are desired, depending on the biologists’ goal, e.g. high production or cell localization of a protein, respectively. To date, however, quantitative information about the expression strength of a promoter is difficult to obtain due to the lack of noninvasive and quick approaches to measure the levels of RNA and protein in cells.

Here, we engineer a fluorescence-based sensor that can provide information on both transcription strength and translation efficiency that is noninvasive, easily applied to a variety of promoters, and capable of providing results in a time frame that is short when compared to current technologies. The sensor is based on the use of an RNA aptamer (termed Spinach) and a fluorogen activating protein (FAP). Both the Spinach and FAP become fluorescent in response to binding with dye molecules. The combination of FAP and Spinach will allow us to quantitatively determine relationships involving mRNA and protein, such as translational efficiency.

To demonstrate the utility of the sensor, we will construct and characterize several T7Lac promoters. For each of the promoters, we will measure the mRNA and protein fluorescence during synthesis and after the synthesis ceased as a function of the concentration of dyes added to the cells. The time dependent fluorescence measurements of mRNA and protein levels will be used in a model that allows one to calculate two important characteristics of gene expression, namely the polymerase per second (PoPS) and translational efficiency. Information about other characteristics of the cell, such as degradation constants for mRNA and protein, and transcriptional efficiency, will be obtained indirectly.

The outcome of this project will consist of a family of promoters, whose strength is measured by the newly developed sensor, which covers a relatively broad range of strengths.

Human Practices

The realization of the potential of synthetic biology depends on the number and quality of scientists making significant contributions to the field. Hence, we plan to contribute to raising the awareness of high school students, who may become future scientists, of the interdisciplinary field synthetic biology and of the preparation one needs to become a synthetic biologist.

Specifically we will give several presentations about synthetic biology (including the iGEM competition) and our project to high school students enrolled in the Summer Academy of Math and Science. To bridge the natural intellectual gap between the background of a high school student and the complexity of our project, we will build and use in the demonstrations an affordable, microcontroller-based, hardware platform and associated, open-source, digital simulation software. The combined hardware/software platform will allow the students to directly manipulate electronic components, which are formal equivalents of the Biobricks used in building our sensor, to affect the current and/or voltage, which are formally the equivalent of the PoPs and translational efficiency measured with the sensor. The software, which is based on the same model we create for the analysis of the sensor, will ensure that the data generated by the students is physiologically accurate.

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Promoters

Promoters are upstream sequences that regulate transcription. Promoters are usually short sequences and act as binding sites for a variety of different RNA polymerases. Promoters have different binding affinities based on their sequence and can be characterized in a matter of different ways. Our project looks to measure some of these properties using fluorescence measurements.

What is fluorescence, exactly?

Fluorescence is a property of some molecules, particularly aromatic organic dyes that absorb photons at a certain wavelength and emit them at a longer, lower energy wavelength.

Fluorescence is described using quantum mechanics principles and organic chemistry. Five and six-member rings tend to fluoresce brightly because of electron delocalization and the properties that are associated with electron delocalization through the p-orbitals. Fluorescent molecules come in a variety of flavors and uses based on their properties, and shape. Fluorescent molecules are known as fluorophores and can take the form of organic dyes or proteins. So far, many different types of fluorophores have been discovered, developed and studied in great detail. Typically, fluorescent proteins have a fluorophore that consists of a few side chains that react and form a complex similar to that of an organic dye. For example, GFP (the most common fluorescent protein) has an HBI fluorophore. Our Spinach construct binds to a dye that derives from this fluorophore. Fluorescence of a molecule can depend on conformation, in the case of our fluorogen, malachite green, which is a conditional fluorophore, the molecule must be in a certain conformation to fluoresce, otherwise, it will absorb photons, but it will emit them very inefficiently (extremely low quantum yield). Fluorescence is a widely studied phenomena and a lot of research is involved with improving current fluorescence technologies and its applications.

What is Spinach?

Spinach is an RNA sequence that can be expressed in cells (in this case, E. coli ) and fluoresces green when DFHBI (an organic dye) is bound to it that can be used to quantify RNA concentration in a cell. Spinach binds to an organic dye called DFHBI which doesn't fluoresce by itself but fluoresces very brightly when it is bound to Spinach. DFHBI is chemically derived from the chromophore in GFP but is altered to increase brightness when bound to RNA. Other fluorescent RNAs have been described but many are non-specific and have many unwanted functions like cytotoxicity. Other fluorescent RNAs also are difficult to quantify because the cells' RNAses (RNA destroying enzymes) can cut out the fluorescent sequence at unpredictable times, making quantification impractical. Spinach utilizes a scaffold that derives from a tRNA sequence, which disguises the RNA so that RNAses leave it alone. Manipulations to the sequence that Spinach is attached to allows for a variety of analyses functions. RNA can be arranged to bind to just about any small molecule in the same way that Spinach was developed (using a SELEX method) to track cellular metabolites. This allows for quantification of another important system in cells. However, in our system, Spinach is incorporated in the mRNA (between the promoter and the RBS) so mRNA is quantified. Click for more information on Spinach. Spinach is the first published RNA sequence of its kind and more sequence/dye combinations are in development; as a result, in years to come, multiple genes (both RNA and protein) can be analyzed in great detail simultaneously.

What is a FAP?

A fluorogen activating protein is a small (26-35kD) protein that derives from the variable region in an antibody. FAPs are not fluorescent unless a fluorogen (also not normally fluorescent) is added, in which case the FAP changes the conformation of the fluorogen and the complex fluoresces brightly. FAPs are currently used to tag certain proteins like actin or tubulin in mammalian cells. FAPs are not primarily expressed in E. coli although we have expressed certain FAPs in E. coli. The two main dyes that the current series of FAPs bind to are malachite green and thiazole orange; our construct uses a variant that binds to malachite green. These dyes are normally cell impermeable but can be designed to penetrate cell membranes. As a result, they were originally used to tag surface proteins. FAPs are excellent reporters because they are small proteins that are soluble and have virtually no maturation time and are highly photostable unlike traditional variants of GFP. FAP technology is widely unexplored but shows promise for new fluorescent technology. FAPs have been used to track individual molecules to 5nm definition as opposed to the typical 200nm. Engineered dyes, called dyedrons, have been developed that increase fluorescence intensity and can allow researchers to improve on live cell imaging techniques. FAPs are genetically different and respond to different excitation wavelengths so researchers can image multiple proteins at the same time in order to understand complex biological processes.

Why is this project important?

• The ability to monitor protein production with fluorescence is a growing field that promises advances in drug development and improving quality control in drug manufacturing.

• Promoter strength directly affects a cell's ability to perform typical functions like divide or move. Designing a genetic circuit that will not overload the cells is key in synthetic biology.

• Inducible promoters are widely used in synthetic biology but many are under-characterized.




Primary Objective: A New Set of Well-Characterized Promoters



Fluorescence Microscopy

Our primary objective is to develop a series of BioBricks that are well characterized based on our methods of measurement.

We assert that our new method of analyzing promoters can quantify certain properties such as:

1. Translational efficiency in vivo
2. in vivo transcription rates
3. Promoter strength
4. In vivo mRNA and protein half-lives in real time

Secondary Objective: Humanistic Practice

FAQ/Terminology in engineering Escherichia coli to monitor these variables via fluorescence. Find out more about Carnegie Mellon: (CMU Home Page).

As part of our project, we seek to intrigue high school students about synthetic biology and engineering. In this pursuit, we developed an electrical analog of our BioBricks (with a simulated microscope using LEDs and a photoresistor) to teach high school students about:

1. Biological systems and synthetic biology
2. Teamwork in research
3. The interdisciplinary nature of synthetic biology
4. Challenges in interdisciplinary work and how teams overcome these boundaries
5. Gene expression and the central dogma of molecular biology
6. How our BioBrick can be used as a measurement system
7. How scientists tackle real-world problems using an interactive simulation that demonstrates the use of our BioBrick and synthetic biological principles

The Team

The 2012 Carnegie Mellon University iGEM team consists of students from Biology, Electrical and Computer Engineering, Biomedical Engineering and Chemical Engineering.

• Peter Wei
• Yang Choo
• Jesse Salazar
• Eric Pederson
Advisors for the team are from the Chemistry, Biomedical Engineering, Electrical and Computer Engineering, Computational Biology, and Biology departments.

Further Considerations

In the pursuit of our project, as well as the biological aspects, we:

• Considered aspects of scale-up, including the ethical, legal and social implications of our BioBrick,
• Programmed a new piece of software for modeling our BioBrick to students,
• Developed and tested techniques for measuring translational efficiency and transcriptional strength,
• Participated in human practices demonstration and modeled our biological system using a programmable and interactive, electrical analog.
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