Light-regulated Gene Expression in Bacteria : Fundamentals, Advances, and Perspectives

Numerous photoreceptors and genetic circuits emerged over the past two decades and now enable the light-dependent i.e., optogenetic, regulation of gene expression in bacteria. Prompted by light cues in the near-ultraviolet to near-infrared region of the electromagnetic spectrum, gene expression can be up- or downregulated stringently, reversibly, non-invasively, and with precision in space and time. Here, we survey the underlying principles, available options, and prominent examples of optogenetically regulated gene expression in bacteria. While transcription initiation and elongation remain most important for optogenetic intervention, other processes e.g., translation and downstream events, were also rendered light-dependent. The optogenetic control of bacterial expression predominantly employs but three fundamental strategies: light-sensitive two-component systems, oligomerization reactions, and second-messenger signaling. Certain optogenetic circuits moved beyond the proof-of-principle and stood the test of practice. They enable unprecedented applications in three major areas. First, light-dependent expression underpins novel concepts and strategies for enhanced yields in microbial production processes. Second, light-responsive bacteria can be optogenetically stimulated while residing within the bodies of animals, thus prompting the secretion of compounds that grant health benefits to the animal host. Third, optogenetics allows the generation of precisely structured, novel biomaterials. These applications jointly testify to the maturity of the optogenetic approach and serve as blueprints bound to inspire and template innovative use cases of light-regulated gene expression in bacteria. Researchers pursuing these lines can choose from an ever-growing, versatile, and efficient toolkit of optogenetic circuits

Utilizing light as an input and output for synthetic biology and metabolic engineering

Advances in synthetic biology are driven by the synergistic relationship between molecular tools and corresponding experimental readout technologies. Molecular tools allow scientists to perturb or control cells with user-defined inputs, while readout technologies produce measurable outputs from cellular processes. A valuable use case of synthetic biology is in metabolic engineering, where microbes are designed to produce commodity chemicals, fuels, or therapeutics from renewable feedstocks. However, current molecular tools and readout technologies are limited for metabolic engineering. In this thesis, I first expand the molecular toolkit with systems that are designed to maximize function in bioproduction settings. Second, I advance a powerful readout technology, stimulated Raman scattering, to track production in metabolically engineered cells. The through line between both aspects of this work is the medium used to interact with cells: light. Optogenetic regulation of metabolism is an attractive approach because it enables dynamic control, which can mitigate factors that limit production such as product toxicity and metabolic flux imbalances. Yet, most optogenetic tools in E. coli, a common chassis for bioproduction, operate through transcriptional mechanisms that do not function well in high-cell density bioproduction settings. In the first part of this thesis, I introduce novel mechanisms of post-translational optogenetic control through a modular light-inducible protein degradation tag, called LOVtag, as well as a method of creating and screening split-protein libraries. In the second part of my thesis, we utilize light as a readout to extract metabolic information. Specifically, we use stimulated Raman scattering (SRS) imaging to measure free fatty acid production in E. coli. We demonstrate that SRS is a promising technique to study metabolically engineered strains with single-cell resolution, longitudinal tracking, and chemical specificity. In sum, the work in this thesis uses light to both control and measure metabolism

Characterizing the function of the Rv3218 gene in Mycobacterium tuberculosis.

Masters Degree. University of KwaZulu-Natal, Durban.Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a primordial affliction that continues to torment humankind since its known history and prehistory. TB is among the major causes of ill-health and death in the world with an estimated 1.8 million cases of death recorded yearly. The situation is worsened by the emergence of the strains of TB that are regarded as resistant. Recently, Mycobacterium bovis bacillus Calmette-Guerin (BCG), has been the only available vaccine for TB. An intense understanding of Mtb’s biology, should reveal new perceptions that can lead to the improved treatment, diagnostics, vaccines and highly needed control measures. Throughout infection, Mtb produces some proteins into the host environment to play critical role in pathogen host interactions. Close to half of the Mtb genome consists of genes with unknown functions. Among those genes is Rv3218 gene which was identified in the study by Chiliza et al., 2019. The Rv3218 gene is hypothesised to have a Diacylglycerol kinase activity. This study aimed at characterising the function of Rv3218 gene in Mtb with the purpose of coming up with ideas of how that can be used in the development of more effective and convenient diagnostic tools, therapeutics, or the total elimination of TB. There is a vast amount of molecular techniques that are currently used to characterise unknown genes. Here we employed a CRISPRi dCas9 system for the silencing of the Rv3218 gene in Mtb. We also used a number of Bioinformatics tools for in silico analysis of the gene and construction of all relevant primers necessary for this molecular cloning. The Rv3218 knockdown repressed by Anhydrotetracycline (ATc) was constructed for assaying the effect of this gene silencing compared to the MtbH37Rv wild type. We then conducted growth curves and MICs (Minimum Inhibitory Concentrations) to check if this gene has an impact on antimicrobial susceptibility and growth of Mtb. We also tested its activity as a diacylglycerol kinase via osmolarity assay as it is said that dgk mutants do not grow well on nutrient media of low osmolarity. On bioinformatics analysis, we found that the gene has cell wall and transcription regulatory functions and possesses a similar structure as diacylglycerol kinase. However, the in vitro analysis was contradictory to these findings. We found that the Rv3218 gene has no impact on the growth of Mtb and it’s susceptibly to the antimicrobial drugs that were used in this study. On the osmolarity assay, there was no observable difference between the growth of the wild type and the knockdown strain in all the concentrations of osmolarity. Judging from these findings, we then concluded that this gene does not function as a diacylglycerol kinase. We then suggested that, more advanced experimental studies still need to be conducted in order to confirm this hypothesis as we were unable to do them due to the short time frame for this study

Exploiting natural chemical photosensitivity of anhydrotetracycline and tetracycline for dynamic and setpoint chemo-optogenetic control

The transcriptional inducer anhydrotetracycline (aTc) and the bacteriostatic antibiotic tetracycline (Tc) are commonly used in all fields of biology for control of transcription or translation. A drawback of these and other small molecule inducers is the difficulty of their removal from cell cultures, limiting their application for dynamic control. Here, we describe a simple method to overcome this limitation, and show that the natural photosensitivity of aTc/Tc can be exploited to turn them into highly predictable optogenetic transcriptional- and growth-regulators. This new optogenetic class uniquely features both dynamic and setpoint control which act via population-memory adjustable through opto-chemical modulation. We demonstrate this method by applying it for dynamic gene expression control and for enhancing the performance of an existing optogenetic system. We then expand the utility of the aTc system by constructing a new chemical bandpass filter that increases its aTc response range. The simplicity of our method enables scientists and biotechnologists to use their existing systems employing aTc/Tc for dynamic optogenetic experiments without genetic modification.ISSN:2041-172