Promoters and Reporters in Synthetic Biology

What Are Promoters and Reporters?

Promoters and reporters are genes used in engineering gene circuits. Promoters are DNA sequences located upstream of a gene; they provide binding sites for transcription factors and, eventually, RNA polymerase, allowing genes to be transcribed. If a promoter is being repressed, then transcription cannot occur as RNA polymerase will not have a place to bind.

Reporters are not as specific as promoters; they are genes that convey some easily-identifiable and measurable characteristic when they are transcribed, such as fluorescence or beta-galactoside proteins. Reporters are generally attached to other gene sequences so the scientist has a way of knowing if the gene is being transcribed - if the reporter is being transcribed, one can assume that the gene of interest is being transcribed as well.

Synthetic, Artificial, and Mutated Promoters and Reporters

Directed evolution is often used to mutate promoters or reporters in order to obtain desirable attributes. Directed evolution of a gene or protein sequence generally mutates or scrambles the sequence in question, screens it for a certain mutation (any cell not displaying the desirable phenotype is removed), and then amplifies the surviving cells so that the process can begin again. However, directed evolution does not produce a truly random promoter library; it can only produce mutated versions of an existing promoter. While this can be useful if only a slightly different version of an existing promoter is needed - say, a faster-degrading GFP - it cannot synthesize a promoter de novo.

In that case, combinatorial promoters can be synthesized as in Cox, Surette and Elowitz (2007). In their experiment, Elowitz et al designed modular sequence units corresponding to the three coding segments of a promoter gene. These segments, assembled at random, can create a diverse and new promoter library that can then be specified via directed evolution.

See Figure 1 for a diagram of combinatorial promoter synthesis.

Random assembly ligation generates a diverse promoter library. Promoters can be assembled out of modular sequence units. (A) The assembled sequence of an example promoter. The 5' overhangs of each unit are shown in red. The RNA polymerase boxes (-10 and -35) are highlighted in yellow, and the predicted start site of transcription (+1) is capitalized. Operator colors are consistent throughout the figure. (B) Steps in promoter assembly and ligation into the luciferase reporter vector: promoters are assembled by mixed ligations using 1-bp or 2-bp cohesive ends, and then ligated into a luciferase reporter plasmid. (C) Luminescence measurements in 16 inducer conditions ( each of four inducers, as indicated) for the promoter shown in (A). The output levels determine promoter logic. Note that this promoter does not respond to LuxR regulation at the distal region. (D) The 48 unique units used in the library contain operators responsive to the four TFs (indicated by color) in the regions distal, core, and proximal. [1]

Why use synthetic/mutated promoters and reporters?

Since much of synthetic biology is based on modeling genetic and molecular mechanisms before they are built, a scientist has to be able to predict how the components of a mechanism or gene circuit will work in order to predict how the whole mechanism will work. Because they have been specifically designed and selected for, synthetic promoters and reporters are easier to predict and model. See Rosenfeld, Young, Alon, Swain, and Elowitz (2007)for more on predicting activity based on components.

Of course, the noise and randomness inherent in cellular interactions mean that no promoter or reporter's activity can be perfectly predicted.

Also, synthetic promoters and reporters are useful for when a wild-type promoter or reporter is not sufficient or lacks some property necessary for a cellular mechanism to work. For example, a reporter protein such as GFP does not degrade as soon as it is produced, so in any mechanism that has to detect a transient signal, GFP would not be a useful reporter. However, a mutated GFP, which degrades faster or in the presence of a certain compound, would negate this effect. The same principle applies for reporters which are more active at lower-than-normal or higher-than-normal temperatures. See Patterson GH et al (1997).

Works Cited

• Weiss R, Basu S, Hooshangi S, Kalmbach A, Karig D, Mehreja R, and Netravali I (2003). Genetic circuit building blocks for cellular computation, communications, and signal processing. Natural Computing 2 (1). Epub 2004 November 02. Abstract

• Arnold FH (1997). Design by Directed Evolution. Acc. Chem. Res.,31 (3). Epub 1998 February 28. Full Text