Team:Carnegie Mellon

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Carnegie Mellon iGEM 2012


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Quantitative In Vivo Promoter Characterization Using Fluorescent Biosensors

Using fluorescent technology to develop new promoters



Image:Cmu2.jpeg

 

Contents

Introduction: Motivation and Background

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.

And Introduction to 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. In our case, we are characterizing promoters that bind to RNA polymerase from the T7 phage. The T7 RNA polymerase binds to its promoter very tightly and produces a high amount of expression. The lac operator is a short sequence that binds to the LacI repressor, which prevents transcription. The LacI protein responds to lactose in the cell. Lactose analogs have been made which are not consumed by E. coli and "turn on" the gene of interest. Our promoters have different affinities to the T7 RNA polymerase and the LacI repressor and therefore have different properties.

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

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?

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?

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