Transcriptional regulation (CarH)
Issues with current bacterial optogenetic transcription regulators.
Most of the bacterial optogenetic tools are initially created for eukaryotic systems and then transferred to prokaryotic cells. This principle creates a suboptimal design for bacterial optogenetic transcription regulators as the eukaryotic and prokaryotic transcriptional apparatuses are fundamentally different.
Eukaryotic optogenetic transcription regulators work because, in the light excited state, they have an increased affinity towards DNA. When the regulatory protein gets bound to DNA, it brings the transcription factor to the promoter and induces transcription. However, the same principle does not work well in bacterial transcription regulation mainly because, in the uninduced state, no protein represses promoter. Such systems show a high basal expression level and low dynamic range between the induced and the uninduced states, making it difficult to apply these systems to synthetic biology.
Other optogenetic transcription regulators are developed from two-component bacterial systems. Even though these tools have up to a few hundred times higher dynamic range, two-component systems also have some disadvantages.
The problem is that two-component systems require a considerable amount of cellular resources – the cell must produce two different proteins for signal transduction, and there are additional genes for chromophore synthesis 1. That is why the cell lacks space for light controllable proteins. In addition to this, signals are relatively slow. All these features make two-component systems complicated for application in more complex circuits.
CarH: for the design of a one-component repressor-based system.
The perfect design of bacterial transcription regulators is a one-component repressor-based system with low leakage and high dynamic range.
All these features are present in the CarH protein from Thermus thermophilus. This protein has cobalamine (Vitamin B12), which has a photosensitive Co-C bond, as a prosthetic group. In the dark, CarH is a tetramer that binds strongly to DNA and represses protein translation 2. When excited by green light, the photosensitive Co-C bond breaks, this disrupts the tetramer structure, and then gene expression can start.
There is no surprise that the CarH protein shows excellent results as an optogenetic tool in eukaryotic systems inducing protein expression 350-fold 3. However, there are no scientific articles on CarH as a bacterial optogenetic tool.
For our experimental procedure, we have constructed a plasmid carrying a CarH cassette under a constitutive Anderson promoter, and an RFP cassette under the CarH inducible promoter of our design. We used a constructed plasmid to check if our system is a viable optogenetic tool in E. coli cells.
The first question to tackle was whether it is better for the CarH gene to be under a strong (Part:BBa_J23100) or a weak (Part:BBa_J23114) consecutive Anderson promoter. The results show that the system works better with the CarH gene under a weak promoter. The experiment was conducted with 20 μM cyanocobalamin, the cells were grown in 30 degrees celsius overnight. With CarH gene under a weak promoter, we were able to observe a 45 fold difference between bacteria grown in light and in the dark and a 2 fold difference with the gene under a strong promoter. Therefore, the weak Anderson promoter version is denoted as mRFP1-CarH device (strong) to and the strong Anderson promoter version is denoted as mRFP1-CarH device (weak).
Figure 1. A graph showing the difference in mRFP expression between CarH device containing E. coli grown in the dark and in the light. Bacteria were grown in 30 °C for 14 hours, TG1 strain
The underlying cause of this observation may be the fact that a lower level of the CarH protein allows easier and more effective required oligomerization.
An experiment was designed to see, if the CarH sysem is modular (protocol). An overnight culture (without cobalamin) was diluted with liquid LB media (containing 10 μM cobalamin), thus allowing the CarH protein to repress in the dark state. The bacteria were kept varying amounts on time in the dark and later brought to light and we were able to observe a change in fluorescence divided by OD 600. The bacteria were grown in 30°C.
Figure 2. An examplary graph showing how the fluorescence divided by OD 600 changes in the same bacteria containing tube when it is brought from the light to the dark and then again to the dark. The last state the bacteria were in before measuring is denoted as the colour of the point, dark blue point indicates the dark and the light orange poitn denotes the light. TG1 E.coli strain grown in 30°C
1. Chatelle C, Ochoa-Fernandez R, Engesser R, et al. A Green-Light-Responsive System for the Control of Transgene Expression in Mammalian and Plant Cells. ACS Synth Biol. 2018;7(5):1349-1358. doi:10.1021/acssynbio.7b00450
2. Padmanabhan S, Pérez-Castaño R, Elías-Arnanz M. B12-based photoreceptors: from structure and function to applications in optogenetics and synthetic biology. Current Opinion in Structural Biology. 2019;57:47-55. doi:10.1016/j.sbi.2019.01.020
3. Schmidl SR, Sheth RU, Wu A, Tabor JJ. Refactoring and optimization of light-switchable Escherichia coli two-component systems. ACS Synth Biol. 2014;3(11):820-831. doi:10.1021/sb500273n
Transcriptional regulation (BphP1)
Near-infrared (NIR) light is of high pursuit for optogenetic regulators due to its low phototoxicity and its spectral isolation from most of the currently known optogenetic tools. What is more, NIR light falls into the near-infrared window in biological tissue, thus has a huge potential to be applied for the control of bacteria inside a mammalian organism.
Noninvasiveness, high activation rates, fast reversibility, and absence of possible side effects would make a perfect system for microbiota control, research of biofilm formation inside a living organism, or for bacterial cancer therapy for very specific protein pharmaceuticals release in a tumor. Most of the phytochromes undergo photoconversion in the range of 660-700nm. However, there is a small class of phytochromes which senses undergo 740-780nm.
For our experiments, we chose bacteriophytochrome photoreceptor 1 (BphP1), which is found in various purple photosynthetic bacteria senses 740-780 nm NIR light switching to the biologically active Pfr conformation. BphP1 binds to and inhibits the transcriptional repressor PpsR2 in this way, activating transcription of photosystem promoters.
BphP1-PpsR2 has already been used for the development of the optogenetic system for mammalian cells and used for targeting proteins to the nucleus or plasma membrane.
Later on, the 40-fold contrast of light-induced gene expression was demonstrated in cultured cells.
The same system was applied for engineering near-infrared light sensor in E. coli. However, even after a thorough optimization, the best received dynamic range between induced and uninduced states was 2.5 times.
Such a range is too low for any practical application. Therefore, together with the team, we decided to develop an optogenetic system with a higher dynamic range.
To improve this system's dynamic range, we decided to try truncating PpsR2.
PpsR2 consists of N-Pas1, Q-linker, Pas1, Pas2, and HTH domains. Q-linker is a dimerization domain. Its structure is disrupted by Pfr state BphP1. Therefore, we decided to fuse Q linker to two different DNA-binding domains: Gal4 from S. cerevisiae and LexA from Bacillus subtilis. We also tried linkers of different length between LexA DNA-binding domain and Q-Pas1.