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Quantitative In Vivo Promoter Characterization Using Fluorescent Biosensors

Using fluorescent technology to analyze new promoters

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.

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.

Primary Objective: A New Set of Well-Characterized Promoters

Fluorescence Microscopy

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. The iGEM competitions and how iGEM works
2. Biological systems and synthetic biology
3. Teamwork in research
4. The interdisciplinary nature of synthetic biology
5. Challenges in interdisciplinary work and how teams overcome these boundaries
6. Gene expression and the central dogma of molecular biology
7. How our project measures properties of promoters
8. How synthetic biologists tackle real-world problems

The Team

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

• Peter Wei (ECE, BME)
• Yang Choo (ChemE, BME)
• Jesse Salazar (ECE, BME)
• Eric Pederson (Bio)

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.