Method for regulating and controlling polygene expression through frequency amplitude and duty ratio of single input signal

Abstract

The invention provides a method for regulating and controlling polygene expression through single input signal frequency illumination amplitude and duty ratio. The method comprises the following steps: S1) constructing an engineering microorganism strain, wherein the engineering microorganism strain comprises a signal response module, a transcription activation module and a multi-target gene parallel expression module; s2) performing signal regulation and control by utilizing different frequencies, duty ratios and illumination amplitudes of the photostimulation signals to realize expression regulation and control of the target gene; and S3) performing quantitative detection on the expression product of the target gene. Compared with the prior art, the method has the advantages that a plurality of downstream genes can be controlled at the same time through single input by imitating the second messenger principle, that is, quantitative expression of a plurality of products can be realized by utilizing a single structure, and quantitative data of different target genes are more comparable while the operation is simple.

Description

Technical Field The present invention belongs to the field of bioengineering, and particularly relates to a method for regulating multi-gene expression by controlling the frequency, amplitude and duty cycle of a single input signal. Background Art Microbial technology aims to produce various valuable chemical products through biocatalysis by microorganisms such as bacteria. However, the expression of heterologous synthetic pathways in microbial cells has an adverse effect on bacterial growth and gene expression. This is due to reasons such as the toxicity of pathway intermediates, coenzyme imbalance, and uneven distribution of bacterial resources. In order to maximize the production of chemical products while ensuring a high growth density, it is necessary to dynamically control the expression of key genes or pathways in microbial cells. Cells initiate gene expression by responding to specific signals, thus achieving localization in the environment, communication, and constructing complex patterns. Engineers attempt to utilize this ability to program cells to perform tasks or produce chemical substances and materials that match the complexity of nature [1]. When currently designing cells to express target products, generally a single signal is used to regulate a single expression circuit to achieve quantitative expression. If multiple products need to be quantitatively expressed simultaneously, traditional methods require designing multiple independent expression circuits that do not interfere with each other. This design places a great burden on bacterial cells, requires a large amount of energy to support the operation of numerous circuits, and at the same time, it is difficult for complex circuits to accurately represent the actual situation inside bacterial cells.

[0001] Brophy, J., Voigt, C. Principles of genetic circuit design. Nat Methods 11, 508–520 (2014). https: / / doi.org / 10.1038 / nmeth.2926 Summary of the Invention When the prior art realizes the synchronous quantitative expression of multiple products, it is necessary to design a large number of repetitive and complex independent expression circuits, which brings a great burden to bacterial cells. In addition, these circuits are difficult to accurately reflect the actual situation inside the cells, and the regulation effect is also difficult to quantitatively evaluate. The present invention proposes to use a single-input regulation method, which can achieve fine synchronous regulation of multiple horizontal expression circuits. The present invention provides a novel single-signal regulation method, which can more flexibly and controllably dynamically control the quantitative expression of key genes or pathways in microbial cells. The present invention proposes to use a theoretical model to guide verification to solve the key technical bottleneck problem of controlling multi-gene quantitative expression in microorganisms with a single input. One aspect of the present invention provides a method for controlling multi-gene quantitative expression in microorganisms with a single-signal input, the method comprising the following steps: S1) Construct an engineered microbial strain: The engineered microbial strain includes a signal response module, a transcriptional activation module, and a multi-target gene parallel expression module; The signal response module is a system composed of protein molecules that control the generation and degradation of multi-gene quantitative expression signal molecules; the genes of the protein molecules that control the generation and degradation of multi-gene quantitative expression signal molecules are knocked out in the engineered microbial strain, and the genes encoding the protein molecules that control the generation and degradation of multi-gene quantitative expression signal molecules are transcribed into plasmids, and the plasmids are transferred into the engineered microbial strain, and the protein molecules that control the generation and degradation of multi-gene quantitative expression signal molecules are quantitatively expressed; the protein molecules that control the generation and / or degradation of multi-gene quantitative expression signal molecules are activated or inhibited by light stimulation signals; The transcription activation module includes multi-gene quantitative expression signal molecules and transcription regulatory proteins; the concentration of the multi-gene quantitative expression signal molecules is regulated by the signal response module, the transcription regulatory proteins can form a complex with the multi-gene quantitative expression signal molecules, and the complex can regulate the parallel expression of multi-target genes in the multi-target gene parallel expression module; the genes of the transcription regulatory proteins in the engineered microbial strain are knocked out, and the genes encoding the transcription regulatory proteins are transcribed into plasmids, and the plasmids are transferred into the engineered microbial strain; The multi-target gene parallel expression module contains more than one target gene, and the target genes have independent promoters; S2) Use light stimulation signals with different frequencies, duty cycles, and illumination amplitudes for signal regulation to achieve the expression regulation of target genes; S3) Quantitatively detect the expression products of target genes. Furthermore, in the system composed of protein molecules that control the generation and degradation of multi-gene quantitative expression signal molecules, the multi-gene quantitative expression signal molecule is cAMP, the protein molecule that controls the generation of multi-gene quantitative expression signal molecules is bPAC, and the protein molecule that controls the degradation of multi-gene quantitative expression signal molecules is CpdA. Furthermore, the light stimulation signal is a blue light signal. Furthermore, the transcription regulatory proteins are Vfr and Crp. Furthermore, the engineered microbial strain is selected from Escherichia coli and Pseudomonas aeruginosa. Furthermore, the frequency range of the light stimulation signal is 0.0001 - 1 Hz, the illumination amplitude is 0.1 - 10000 μW / cm 2 and the duty cycle range is 0.1% - 99.9%. Furthermore, the target genes are genes encoding fluorescent proteins and active proteins. Furthermore, it further includes step S4), analyzing that there are multiple steady states in the expression of target genes under the input of combined parameters of different light stimulation signal frequencies, duty cycles, and illumination amplitudes. Further, it further includes step S4), and realizes the controllable switching of downstream genes among multiple steady states by adjusting the combined parameters of the light stimulation signal frequency, duty cycle, and light intensity. Further, it further includes step S4), and generates the mapping relationship between the combined parameters of the light stimulation signal frequency, duty cycle, and light intensity and the expression state of downstream genes. Further, it further includes step S4), and obtains the state space of the downstream gene steady state by detecting the expression level. Beneficial effects 1) Compared with the prior art, the present invention can, by imitating the second messenger principle, achieve the simultaneous control of multiple downstream genes by a single input, that is, it can use a single structure to achieve the quantitative expression of multiple products. While the operation is simple, the quantitative data of different target genes are more comparable. 2) By continuously changing the signal for quantitative regulation of each circuit, it is more flexible and precise than the traditional switching method. 3) Real-time regulation of multiple processes, without the time delay caused by the interval stage of the traditional method. Description of the drawings Figure 1 It is the gene circuit diagram of the present invention. Figure 2 It is the time-concentration kinetic simulation diagram of cAMP and multiple fluorescent proteins. Figure 3 It is the comparison of the state space achieved by the prior art and different methods of the present invention. Specific embodiments In order to make the above objects, features, and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below, but it should not be construed as a limitation on the implementable scope of the present invention. In some embodiments of the present invention, a method for controlling the quantitative expression of multiple genes in microorganisms by a single signal input is provided. As Figure 1 shown, it shows a method using the second messenger cAMP as the signal molecule for the quantitative expression of multiple genes and multiple fluorescent proteins as the quantitative signal molecules. The method includes the following steps: S01) Construct an engineered microbial strain: The original expression genes related to bPAC and CpdA in the engineered microbial strain are knocked out, where bPAC is a light-sensitive adenylate cyclase that promotes the conversion of ATP to cAMP molecules, and CpdA is a cAMP phosphodiesterase that degrades cAMP molecules at a constant rate; and the genes encoding bPAC and CpdA are transcribed in a plasmid and transduced into the engineered microbial strain to achieve precise quantification. Since the values of bPAC and CpdA can be precisely regulated, the synthesis and degradation of the corresponding cAMP can be precisely regulated by the external input signal light. The engineered microbial strain also contains a gene and a promoter of a fluorescent protein as a quantitative signaling molecule. The fluorescent protein as a quantitative signaling molecule can be any known fluorescent protein in the art, as long as it can achieve the purpose of quantitative detection. In this specific embodiment, there are 3 fluorescent proteins as quantitative signaling molecules, namely CyOFP, RFP, and sfGFP, respectively. They are controlled by different promoters. The engineered microbial strain also contains a transcriptional regulatory protein. For example, in some specific embodiments of the present invention, the engineered microbial strain is Pseudomonas aeruginosa, and its transcriptional regulatory protein is Vfr. The transcriptional regulatory protein can perform Hill binding with the multi-gene quantitative expression signaling molecule, and the complex formed by the transcriptional regulatory protein and the multi-gene quantitative expression signaling molecule can coordinately regulate the expression of multiple downstream quantitative signaling molecule genes. The original expression gene related to the transcriptional regulatory protein is knocked out, and at the same time, the gene encoding the transcriptional regulatory protein is transcribed in a plasmid and transferred into the engineered microbial strain to achieve precise quantification. In some embodiments of the present invention, the transcriptional regulatory protein can be different according to the type of the engineered microbial strain. For example, the Crp protein can be used as the transcriptional regulatory protein that binds to cAMP in Escherichia coli. S02) Cultivate the engineered microbial strain, in which the plasmid quantitatively expresses bPAC and CpdA, and then stimulate the cells with blue light as a signal source. The blue light can activate the light-sensitive adenylate cyclase bPAC; by controlling the frequency, duty cycle, and light intensity of the input blue light illumination, the expression of multiple downstream quantitative signaling molecule genes can be regulated simultaneously; furthermore, since there are usually multiple downstream genes and the relative strengths of the promoters of different genes are different; by controlling the frequency, duty cycle, and light intensity of the input illumination, the relative ratio of the downstream genes can be precisely regulated. S03) Quantitative detection of the quantitative signaling molecule of the target expression product. As shown in Figure 2 , simulate expression through a plasmid to achieve a quantitative value of 1 μmol for bPAC and 1 μmol for CpdA, a blue light irradiation frequency of 0.01 Hz, a duty cycle of 20%, and a light intensity of 20 μW / mm 2 (at this light intensity, about 1 μmol of bPAC is activated, so a quantitative expression of 1 μmol of bPAC is used to simulate a light intensity of 20 μW / mm 2 ), and the expression of cAMP and three downstream genes. Among them, a frequency of 0.01 Hz means that the cells are stimulated with blue light with a period of 100 s, and a duty cycle of 20% means that in these 100 s, the blue light is irradiated for 20 s and then turned off for 80 s, and so on in a cycle, where the on-time ratio is 20%. Figure 2 The abscissa is time, and the ordinate is the concentration of cAMP and the proteins expressed by three downstream genes. As shown by Figure 2As can be seen from the right figure, the concentration of the fluorescent protein increases rapidly before 3000 s, and after 3000 s, the fluorescent protein reaches a periodic stability, that is, the final equilibrium state. The equilibrium state means that the fluctuation range of the concentration protein is very small, and it can be regarded as a stable state. Integrate and average it, and reduce it to obtain the state value; the integral average value refers to the integral of the expression level with respect to time (within one cycle after equilibrium), and the integral result divided by the cycle is the integral average value. Figure 2 The reason why the final protein fluctuation is very small is that the input period is small. If the input period is large, the final fluctuation will also be large. Taking the data at a single moment as the final value is inaccurate. Therefore, the integral average value within one cycle is used as the final equilibrium state stable value. Reduce the final equilibrium state values of these three fluorescent proteins to the theoretical maximum value (all promoters are activated and transcription and translation are initiated) to obtain a set of data (0.20, 0.09, 0.05), which can be regarded as a state. (0.2, 0.09, 0.05) is a reduced state value, that is, the result of reducing the integral average values of the three proteins to the theoretical maximum value. The three numerical values respectively represent the states of three downstream genes, and the combination of these three numerical values represents a state. The state composed of the above three numerical values can be more intuitively displayed as a point in three-dimensional space. Compare the states that can be achieved by the method of changing the signal intensity in the prior art and the states that can be achieved by the solution of the present invention. The total number of state spaces that can be covered by the traditional method of only adjusting the signal intensity by traversing the illumination amplitude is much smaller than the state space that can be covered by the present invention by adjusting the frequency, adjusting the illumination amplitude, and the duty cycle. By using the above method, by adjusting the frequency range (0.0001 - 1 Hz), illumination amplitude range (0.1 - 10000 μW / cm 2 ) and duty cycle range (0.01% - 99.99%) of the input signal, measure the expression level of the target gene under different parameter inputs. As Figure 3 shown, this is the projection of a three-dimensional cubic dot plot onto a two-dimensional plane. The left figure represents the state space that can be covered by the traditional method of adjusting the illumination amplitude range. Each curve in it represents the state space that can be covered by a strain of bacteria when adjusting the illumination amplitude. The more curved and longer the curve is, the greater the difference in the binding strength of the promoters of the three downstream genes. It can be seen that if it is necessary to design a state space that cannot reach the same initial state, it is necessary to re-culture bacteria for experiments, which is very cumbersome. As Figure 3 shown, Figure 3The left figure below shows the state space that can be covered by the amplitude modulation of three strains. If the required state space is not on this curve, that is, this strain amplitude modulation cannot complete this task, and the strain needs to be redesigned. The key of the present invention is to input the original light amplitude regulation. The method of the present invention adds two parameters when adjusting the light: frequency control and duty cycle control. By adjusting the three parameters of frequency, light amplitude and duty cycle, all the state spaces in the right figure can be achieved in one bacterium, which can save a lot of manpower and material resources. In some specific embodiments of the present invention, the above scheme can also be replaced to meet more applications. For example, the bPAC->cAMP->CpdA system used in the above embodiments can be replaced by other regulatory systems, as long as it satisfies that the multi-gene quantitative expression signaling molecule can be quickly and quantitatively controlled by the light signal to generate and degrade, the cAMP synthesis and degradation system can be replaced. In some specific embodiments of the present invention, the above scheme can also be replaced to meet more applications. Replace the downstream fluorescent molecule with other quantitative signaling molecules. Here, the fluorescent molecule is only used for quantitative analysis and can be transformed into any quantitative molecule. The number of downstream genes can also be 2-10. Different expression states can be distinguished by selecting the strength of the downstream gene promoter. Through the above method of the present invention, the following can be quickly achieved: 1) By adjusting the frequency range (0.0001-1 Hz), light amplitude range (0.1-10000 μW / cm 2 ), and duty cycle range (0.01%-99.99%) of the input signal, measure the expression level of the target gene under different parameter inputs. 2) Analyze that there are multiple steady states in the expression of the target gene under different signal parameter inputs. 3) Through the adjustment of the frequency, light amplitude and duty cycle of the input signal, realize the controllable switching of the downstream gene between multiple steady states. 4) Can quickly find the complex many-to-many mapping relationship between the combination of the frequency, amplitude and duty cycle of the input signal and the expression state of the downstream gene. And obtain the combination of the frequency, amplitude and duty cycle of the input signal with the fastest and most efficient. 5) By regulating the combination of the frequency, amplitude and duty cycle of the input signal, the state space formed only by amplitude regulation can be expanded.

Claims

1. A method for controlling the quantitative expression of multiple genes in microorganisms with a single signal input, characterized in that, The method includes the following steps: S1) Construct an engineered microbial strain: The engineered microbial strain includes a signal response module, a transcriptional activation module, and a multi-target gene parallel expression module; The signal response module is a system composed of protein molecules that control the generation and degradation of a signal molecule for multi-gene quantitative expression; the genes of the protein molecules that control the generation and degradation of the signal molecule for multi-gene quantitative expression are knocked out in the engineered microbial strain, and the genes encoding the protein molecules that control the generation and degradation of the signal molecule for multi-gene quantitative expression are transcribed into a plasmid, and the plasmid is transferred into the engineered microbial strain, and the protein molecules that control the generation and degradation of the signal molecule for multi-gene quantitative expression are quantitatively expressed; the protein molecules that control the generation and / or degradation of the signal molecule for multi-gene quantitative expression are activated or inhibited by a light stimulation signal; The transcriptional activation module includes a signal molecule for multi-gene quantitative expression and a transcriptional regulatory protein; the concentration of the signal molecule for multi-gene quantitative expression is regulated by the signal response module, the transcriptional regulatory protein can form a complex with the signal molecule for multi-gene quantitative expression, and the complex can regulate the parallel expression of multi-target genes in the multi-target gene parallel expression module; the gene of the transcriptional regulatory protein in the engineered microbial strain is knocked out, and the gene encoding the transcriptional regulatory protein is transcribed into a plasmid, and the plasmid is transferred into the engineered microbial strain; The multi-target gene parallel expression module contains more than one target gene, and the target gene has an independent promoter; S2) Use signal regulation with different frequencies, duty cycles, and illumination amplitudes of the light stimulation signal to achieve the expression regulation of the target gene; S3) Quantitatively detect the expression product of the target gene.

2. The method for controlling the quantitative expression of multiple genes in microorganisms with a single signal input according to claim 1, characterized in that, In the system composed of the protein molecules that control the generation and degradation of the signal molecule for multi-gene quantitative expression, the signal molecule for multi-gene quantitative expression is cAMP, the protein molecule that controls the generation of the signal molecule for multi-gene quantitative expression is bPAC, and the protein molecule that controls the degradation of the signal molecule for multi-gene quantitative expression is CpdA; Preferably, the light stimulation signal is a blue light signal.

3. The method for controlling the quantitative expression of multiple genes in microorganisms with a single signal input according to claim 2, characterized in that, The transcriptional regulatory proteins are Vfr and Crp.

4. The method for controlling the quantitative expression of multiple genes in microorganisms with a single signal input according to claim 1, characterized in that, The engineered microbial strain is selected from Escherichia coli and Pseudomonas aeruginosa.

5. The method for controlling the quantitative expression of multiple genes in microorganisms with a single signal input according to claim 1, characterized in that, The frequency range of the light stimulation signal is 0.0001 - 1 Hz, the light intensity is 0.1 - 10,000 μW / cm 2 and the duty cycle range is 0.01% - 99.99%.

6. The method for controlling the quantitative expression of multiple genes in microorganisms with a single signal input according to claim 1, characterized in that, The target gene is a gene encoding a fluorescent protein or an active protein.

7. The method for controlling the quantitative expression of multiple genes in microorganisms with a single signal input according to claim 1, characterized in that, It further includes step S4), analyzing that there are multiple steady states in the expression of the target gene under the input of different combination parameters of the light stimulation signal frequency, duty cycle, and illumination amplitude.

8. The method for controlling the quantitative expression of multiple genes in microorganisms with a single signal input according to claim 1, characterized in that, It further includes step S4), realizing the controllable switching of the downstream gene between multiple steady states through the regulation of the combination parameters of the light stimulation signal frequency, duty cycle, and illumination amplitude.

9. The method for controlling the quantitative expression of multiple genes in microorganisms with a single signal input according to claim 1, characterized in that, It further includes step S4), generating a mapping relationship between the combination of the light stimulation signal frequency, duty cycle, and illumination amplitude and the expression state of the downstream gene.

10. The method for controlling the quantitative expression of multiple genes in microorganisms with a single signal input according to claim 1, characterized in that, It further includes step S4), obtaining the state space of the downstream gene steady state by detecting the expression level.

Images