Promoters, initiators of transcription and drivers of synthetic biology

Promoter characterisation and engineering is a common and arguably useful component of many synthetic biology studies. This post covers some recent articles about promoter construction and transcriptional control, and a few thoughts on the future directions and applications.

When engineering a biological system for a specific purpose, one would desire a degree of predictability in the design. As the novel information inserted into the system is usually DNA encoded, while the function is often carried out by RNA or protein molecules, the transcription is usually the point of control: it regulates when, how long, and how much an RNA molecule will be produced. The idea behind well-characterised systems, driven by promoters with known behaviour, crystalizes in the study and generation of genetic circuits and genetic logical gates, one of the most iconic outcomes of synthetic biology—for a detailed outline of synthetic circuits and regulation refer to this excellent review by Bashor and Collins. Despite the plethora of studies on promoter characterisation and the amount of available parts (just browse the iGEM part registry to find hundreds if not thousands of deposited promoters of various degrees of characterisation), precise transcriptional control is not fully achieved in most biological systems, and there are several factors that affect transcription outcome.

Precise control of copy number and induction by small molecules

In an article published in Nature Biotechnology, researchers from Chris Voigt’s lab at MIT reduced the variance of transcripts caused by alternating copy number of the DNA of interest (e.g. when using different self-replicating vector or when integrating a sequence into the genome). The scientists implemented an incoherent feedforward loop (iFFL), which, according to Dr Thomas Segall-Shapiro, the lead author of the article, is “a simple regulatory motif where an input signal (in our system, copy number) affects the output (the gene of interest). The signal is additionally inverted through an intermediary (the TALE repressor) to have the opposite effect on the output”. The precision of control achieved is impressive, and Dr Segall-Shapiro believes that “this design should be applicable to many organisms. However, careful construction and tuning must be done to achieve results as precise as what we saw in E. coli. The key is finding a repressor that is non-cooperative and very strong”.

The same research group investigated another promoter regulation issue in a recently published preprint, the generation of inducible promoters. The resulting “Marionette” strains are able to respond 12 different small molecules, with low background expression and over 100-fold induction. Adam Meyer, the lead author of this work, describes how he envisions the applications of this system: “Our next experiments will explore how Marionette can be applied to quickly optimize the gene expression levels in a biosynthetic pathway. Marionette cells will be used to independently control the expression of multiple enzymes in a pathway, assigning each enzyme to its own sensor. It should be possible to quickly determine the optimal level of each of the enzymes in the pathway using different levels of induction.  This approach would be faster, cheaper, and easier than using more traditional techniques that involve constructing a new DNA molecule for each set of enzyme levels”.

Promoters specific to certain cell types

Promoters that are active in certain cell types or during defined developmental stages can be immensely useful in fundamental research studies and can have potential applications in fields such as precision medicine. In an interesting work published in PLOS ONE, Georgios Pothoulakis and Tom Ellis report yeast promoters that are specific for mother cells that have contributed in daughter cells in a population. Dr Pothoulakis started working on such a system as he was “looking into controlling common yeast phenotypes (such as pseudohyphal growth) and creating cell populations that differentiate using synthetic gene networks. I engineered a hybrid promoter based on the widely known HO that is also inducible and thus could be incorporated into genetic circuits. Conveniently, the HO system is only active in haploid cells, which was always the goal of our studies”.

Interchangable parts and future of promoter research

One of the biggest challenges in synthetic biology parts is the inability to transfer easily to different biological systems that the ones they were generated in. Dr Pothoulakis reflects that “it is true that in many cases we don’t observe the expected behaviour when the same or similar elements are transferred to other organisms. In the synthetic biology field, there is an over-reliance on characterization done in prokaryotes (usually because design is more straightforward and general handling is easier). The parts are then expected to behave similarly to eukaryotes. Usually, we try to overcome this issue by designing our systems to be as orthogonal as possible and following the sequence design rules that the organism of interest dictates. But what we have found is that other factors, like the presence of a nucleus, have an effect on the way several transcriptional elements behave. In other cases, elements that have a dramatic effect in expression in one organism might be overshadowed by others, unique to another type of organism”.

Dr Segall-Shapiro is more worried about the effect of complex regulatory systems: “From my perspective—that is, from one attempting to engineer synthetic genetic systems—the biggest unknown is how different promoters respond to different stress conditions. For example, if we build a genetic program that consumes too many cellular resources, how do the expression levels of all of the promoters in the program and the cell change? And how consistent are these changes among the myriad ways a cell can be perturbed?”

When considering all these limitations, the question whether we are putting “too much” effort in characterising promoters comes to mind. Dr Segall-Shapiro believes that “one advantage of building new regulation into promoters is that it allows the easy combination of such regulation with other genetic elements. Because you are controlling gene expression at the level of transcription, translational or post-translational regulation can be easily be layered on top for further control”. On the other hand, bioengineer Cristiana Dal’Molin from the University of Queensland warns that “while promoters are certainly an important regulatory point, one may overlook elements such as conserved non-coding sequences (CNS) and miRNAs, particularly important for regulation in plants and animals”.

It is true that many non-model organisms do not play by the common rules, while sometimes even bacteria show different relative expression driven by the same promoters but using different reporting proteins. Nevertheless, a promoter is the crucial component of any genetic construct and is the element that initiates the transition from genetic information to cellular function. Therefore, we should expect this sub-field to blossom even more and get enriched with more, tuneable, more accurate, and hopefully transferable promoters to express our genes.