Real-time quantitative measurement of RNA and protein levels using fluorogen-activated biosensors

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.

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 T7/Lac 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.

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Human Practices

The impact of synthetic biology depends on the number and quality of scientists making significant contributions to the field. To this end, we contributed to raising the awareness of high school students, who may become future scientists, about the interdisciplinary field of synthetic biology, and about the preparation one needs to become a synthetic biologist.

We decided to create teaching materials for high school students inspired by our team’s research project. Our goal was that these materials can be easily used by a science teacher in a lecture in a Biology or Chemistry course to (1) explain what Synthetic Biology is, and (2) enable the students to understand how our biosensor works. The teaching materials we have created, specifically a power point presentation and an electronic circuit kit, have become part of the Lending Library of Kits of DNAZone, the outreach program of the Center of Nucleic Acids Science and Technology at Carnegie Mellon. The kits in the Library are loaned to high school teachers in the Pittsburgh area to be used in teaching Math and Science. We have also tested the kit in several demonstrations in the Summer of 2012 to high school students enrolled in the Summer Academy of Math and Science at Carnegie Mellon.

To bridge the gap between the background of a high school student and the complexity of our project, we built an affordable, microcontroller-based, hardware platform and associated, open-source, digital simulation software. The combined hardware/software platform allows the students to directly manipulate electronic components, which are formal equivalents of the BioBricks used in our sensor, and to observe the effect of changing these components on the current or voltage output, which is the equivalent of the fluorescence intensity in our lab experiments. The software part of the platform includes the same model we created for the analysis of the sensor, and the GUI that facilitates the manipulation of the circuit kit.

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Objective 1: Novel Well-Characterized Promoters

Our first 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

The promoters we submit will be characterized with these properties.

Objective 2: Human Practices

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. Synthetic biology and its relationship to Biology and Science and Engineering in general
2. Gene expression and the central dogma of molecular biology
3. How synthetic biologists tackle real-world problems
4. The iGEM competition and how our iGEM team's project enables one to measures the properties of promoters
5. The interdisciplinary nature of synthetic biology
6. The advantages and challenges of interdisciplinary work

Further Considerations

In the pursuit of our project we:

• Considered the ethical, legal and social implications of our BioBrick
• Wrote new software for modeling the performance of our BioBrick
• Developed and tested techniques for measuring translational efficiency and transcriptional strength
• Created materials for teaching high school students about synthetic biology and scientific research. These materials included a programmable and interactive, electrical analog of our biosensor.