An Introduction to Promoters

Promoters are upstream sequences that regulate transcription. Promoters are usually short sequences and act as binding sites for RNA polymerases. Promoters have different binding affinities based on their sequence and can be characterized in different ways. Our project aims 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 RNA. The lac operator is a short sequence that binds to the LacI repressor, which prevents transcription. The LacI protein is inhibited by lactose. Lactose analogs have been made, which are not consumed by E. coli and can be used to "turn on" the gene of interest. Our promoters have different affinities to the T7 RNA polymerase and the LacI repressor and therefore have different measurable properties.

What Is Fluorescence?

Fluorescence is a property of certain molecules, particularly aromatic organic dyes. These molecules can absorb photons at a certain wavelength and emit them at a longer 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 quantum properties that are associated with it. 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, a fluorescent protein has 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 is derived from this fluorophore. The fluorescence of a molecule can depend on conformation, in the case of our fluorogens, malachite green and DFHBI, which are conditional fluorophores, 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. Spinach can be fused to RNAs of interest to quantify RNA concentrations in a cell. Spinach binds to an organic dye called DFHBI that 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. Spinach utilizes a scaffold that derives from a tRNA sequence, which disguises the RNA so that they are less prone to RNases degradation. RNase A has been shown to degrade Spinach, however. 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. In our system, Spinach is incorporated in the mRNA (between the promoter and the RBS). 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 details simultaneously.

What Is a FAP?

A fluorogen activating protein is a small (26-35kD) protein that is derived from a variable region of an human antibody. FAPs do not fluoresce 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 primarily expressed in S. cerevisiae or mammalian cells although some variants have been expressed 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 has great potential for advancing 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. Different FAPs are genetically unique and respond to different excitation wavelengths, hence could be used to image multiple proteins at the same time. Sequences of a few FAPs have been published and they are under patent protection. For this reason, we did not submit our FAPs to the parts registry.

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 cellular functions like divide or move. Designing genetic circuits that do not overload cells is critical for successful implementation of synthetic biological systems.

• Inducible promoters are widely used in synthetic biology and iGEM, but they often lack quantitative measurements of both RNA and protein synthesis rates.