optogenetic control of bacillus subtilis gene expression
The spatial and temporal changes of bacterial gene expression signals are complex.
Although photogenetics tools are ideal tools for studying these processes, none of them are designed for this organism.
Here, we have transplanted a blue bacterial light sensor path containing a green/red photogenic two body
Two component systems CcaSR for the metabolic enzymes used to produce algae blue proteins (PCB)
, And control the output promoter of the gene of interest transcribed into B. subtilis.
In the original
Functional design, We optimized the expression of pathway genes, improved the output of PCB through the translation fusion of biological enzymes, designed a strong chimera output promoter, and increased by a micro photoelectric sensor kinase
Our final design shows more than 70
Folding activation and rapid reaction dynamics to make it
It is suitable for studying a wide range of gene regulation processes.
In addition, the synthetic biology method we developed to link this pathway should make B.
B is a model organism that studies how time changes. varying (dynamic)
, Heterogeneous, and space-
Coordinated control of gene expression signals
For example, in response to short
Energy pressure (i. e.
, Glucose/phosphate limit)
, The transcription of the general stress response regulator is activated in a pulse manner, and the frequency is proportional to the stress intensity.
In contrast, the environment (e. g. , osmotic)
Stress induced a single transcription pulse of this same regulator, whose magnitude is proportional to the start rate of the stress.
In a separate pathway, persistent starvation induced the main regulator of spore formation by a series of pulses with increased amplitude.
Although all cells exhibit these pulses, it may be due to heterogeneity at the master regulator level that only one subset continues to produce spores.
In addition, exciting and noisy genetic circuits drive a small fraction of the cells to briefly differentiate into a state capable of absorbing DNA before spore formation.
From a spatial point of view, a coordinated gene expression pattern can be observed in an undomesticated biofilm, in which moving cells, subsets of matrix cells
Cells distributed in different regions of production and spores.
In addition, metabolism and growth pass through potassium-
The mediated action potential radiating outward from the center.
Although these regulatory dynamics are very rich, potential genetic circuits are usually studied using static and spatial uniform genetic disturbances, including genetic knock-down or chemical-
The induction promoter expresses the genes of interest at different steady state levels.
It is impossible to program artificial gene regulation signals with precise temporal and spatial features, even for the strongest-Research path.
By contrast, engineering lightssensing two-
Component system (TCS)
Abnormal control of gene expression dynamics has been achieved, even at single cell levels.
Similar tools that can accurately control gene expression are needed.
PCC6803 TCS CcaSR is the green light-
Activated transcription regulation pathways (Fig. ).
CcaSR includes blue bacteria (CBCR)-
Family of sensor kinase (SK)
CcaS and OPR/PhoB-
Home response regulator (RR)CcaR.
CcaS contains an assumed N-
The end of the trans-membrane helix, followed by cGMP Diester enzyme/gland-based loop enzyme/FhlA (GAF)
Two for each domainARNT-Sim (PAS)
Domain of unknown function and an his kinase (HK)domain.
CcaS senses light through a linear siyr-producing algae Blue protein (PCB)
, A auxiliary base combined with the GAF domain.
Synthesis of PCB by two steps: Hemin 1 (encoded by )
The oxidation of hemoglobin to Bile Green IX (BV)
, Felidocin redox enzyme (encoded by )
Reduce BV to PCB (Fig. ). Holo-CcaS (hereafter CcaS)
It was produced under the green light (535u2009nm)-
Sensitive ground state with low self-excited enzyme activity.
The absorption of green photons switches CcaS to red light (672u2009nm)-
Sensitive active state with high self-kinase activity.
The active CcaS transfers the phosphate base from ATP to a conserved HK his residue (Fig. )
, Then the conservative gate aspartate residue on CcaR (Fig. ).
The activation of CcaR, which subsequently induced transcription of the P-output promoter (Fig. ).
The red light irradiation restored the active CcaS to the ground state and eliminated it
Possible P transcription via CcaS-activation
De-phosphate mediated by CcaR (Fig. ).
We and others have re-used the CcaSR system as a light genetics tool and used it to achieve abnormal quantitative, spatial and temporal control of gene expression.
In the initial study, we cloned the unmodified genomic site into a vector,
Production of vector transformation by coding the PCB of the synthetic manipulator.
However, this v1.
0 The system displays the leakage output at a red light, and the activation time is less than 5-fold by green. In a follow-
In the study, we reduced the leakage and increased the dynamic range to more than 100
Through system optimization, and-
Operate and truncate P to delete the transcription start point that you do not want (resulting in P).
Sode and his colleagues later constructed several miniature CcaS variants lacking the PAS domain and demonstrated that two of these proteins led to a decrease in P output under red light and a similar P output in green or
We introduce the corresponding CcaS PAS deletion in the context of optimization (v2. 0)
System, causing CcaSR v3.
Very low leakage and nearly 600 system
Fold dynamic range.
Various versions of the CcaSR system have been used separately and are combined with additional light genetics tools with different wavelength specificity to achieve precise temporal and spatial control of one or more gene expressions, including in singlecell level.
In one of the studies, we programmed the linear ramp and sine wave expression of transcription inhibitors to characterize the input/output (I/O)
A broad dynamic
Synthetic gene circuits.
Here we combine the lessons learned from our previous work with several new synthetic biology methods to transplant CcaSR.
Initial design based on CcaSR v2.
0, does not respond to the light.
We use fluorescent protein fusion to reveal this and have poor expression.
The recoding of the initial ORF sequence and several modifications to the gene expression box greatly improved the expression.
Despite these optimizations, we found that the PCB level is still low.
Inspired by previous research on enzyme fusion and scaffolding, we designed-
Pan fusion, which leads to a high level of PCB.
Next, we demonstrate that P from PCC6803 is weak relative to other promoters.
To increase transcription output, we combine P with a powerful constituent promoter.
Then we filter the best-
Perform a miniature CcaS variant in our system.
Finally, we describe the steady state and dynamic I/O of the optimized system CcaSR v1.
0, to prove that it should be able to characterize a wide range of gene regulation processes.
The principles articulated in this debugging and optimization process should be very useful for future synthetic biology applications, and more generally, for any case where a genetic circuit needs to be transplanted between bacterial species.