Promoters and Reporters in Synthetic Biology
Contents
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]
