CN110288076B - Bacterial cell calculation component for indicating priority of signals under different accompanying signals - Google Patents

Bacterial cell calculation component for indicating priority of signals under different accompanying signals Download PDF

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CN110288076B
CN110288076B CN201910613185.6A CN201910613185A CN110288076B CN 110288076 B CN110288076 B CN 110288076B CN 201910613185 A CN201910613185 A CN 201910613185A CN 110288076 B CN110288076 B CN 110288076B
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陈梅
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Minzu University of China
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Abstract

The present disclosure provides a bacterial cell calculation component that indicates the priority of a signal under different concomitant signals. The cell calculation part is composed of a first cell and a second cell, wherein the first cell is used for detecting a signal to be detected and a first accompanying signal, and the second cell is used for detecting the signal to be detected and a second accompanying signal; the gene line in the first cell includes a first line and a second line, and the gene line in the second cell includes a third line and a fourth line. The cell calculation component provided by the invention can make different indications on the simultaneous existence of no signal to be detected, only the signal to be detected, the signal to be detected and corresponding accompanying signals by designing a gene circuit and utilizing the activation of a promoter and the inhibition of an indicating signal, and can indicate a final result by the output combination of multiple cells. The component can be used in biological computers, biological sensors and other related applications which need to judge the priority of a specific signal in different states, and has the advantages of calculation accuracy, high efficiency and usability.

Description

Bacterial cell calculation component for indicating priority of signals under different accompanying signals
Technical Field
The present disclosure relates to the technical field of synthetic biology, and in particular, to a bacterial cell calculation component indicating priority of signals under different accompanying signals.
Background
The construction of cell computing components is one of the hot problems in synthetic biology today. At present, various logic gates, such as cell and/or not, cell computing components, such as bistable switches, oscillators, memories and the like, have been successfully constructed.
Priority is a parameter that determines the priority level at which each task accepts system resources when the computer system is processing a plurality of computing tasks. Cell computers are limited by the computational resources within the cell, and in order for the limited computational resources to be used most efficiently, it is often necessary to compare the importance of the signals, determine their priority, and respond preferentially to high priority signals. Therefore, it is very important for the cell computer to determine the priority. Cell computers operate in complex biochemical environments where the variety of signals varies widely, and for the same signal, different accompanying signals present in the environment, although not directly interacting with it, may have an effect on the priority of its calculation. For example, lactose is essential for escherichia coli when no glucose is present, and becomes optional when glucose is present, the importance changes greatly, and the priority changes accordingly, and at present, the cell computer field does not have a calculation unit for determining the priority of signals under different accompanying signals, and the closest technique is a cell size comparator. However, the conventional cell size comparator can only compare the sizes of three different substances, but cannot compare the sizes of the same substance in different states, thereby indicating the priority. The above problems have become a problem to be solved urgently.
Disclosure of Invention
In order to solve the problems in the prior art, the embodiments of the present disclosure provide a bacterial cell calculating component for indicating the priority of signals under different accompanying signals, which can be used in a biological computer, a biosensor, and other related applications that need to determine the priority of a specific signal under different conditions. The priority indication of the same signal under different accompanying signal states is realized by combining the output results of two or even multiple cells, and the output results are changed by activating a promoter and inhibiting an indication signal, so that the calculation function is realized. The method has the advantages of calculation accuracy, high efficiency and easiness in use.
In a first aspect, the present disclosure provides a bacterial cell calculation component indicating priority of signals under different concomitant signals, the bacterial cell calculation component being composed of two cells, a first cell for detecting a signal to be detected and a first concomitant signal, and a second cell for detecting a signal to be detected and a second concomitant signal;
the gene circuit in the first cell comprises a first circuit and a second circuit;
the first circuit consists of a signal promoter to be detected, a first adjoint signal promoter activating element, a first indicating signal and a terminator in sequence;
the second circuit is sequentially composed of a first adjoint signal promoter, a first adjoint signal promoter suppression element, a first indication signal suppression element, a second indication signal and a terminator;
the gene circuit in the second cell comprises a third circuit and a fourth circuit;
the third circuit consists of a signal promoter to be detected, a second adjoint signal promoter activating element, a first indicating signal and a terminator in sequence;
the fourth line is sequentially composed of a second adjoint signal promoter, a second adjoint signal promoter suppression element, a first indication signal suppression element, a second indication signal and a terminator;
the promoter of the signal to be detected is started by the signal to be detected, the first accompanying signal promoter is started by the first accompanying signal, the second accompanying signal promoter is started by the second accompanying signal, the promoter inhibiting element can inhibit the promoter from working, the promoter activating element can relieve the inhibition of the promoter inhibiting element on the promoter, and the indicating signal inhibiting element can inhibit the expression of the indicating signal.
Preferably, the method further comprises the following steps: when only the signal to be detected exists, the promoter of the signal to be detected in the first circuit of the first cell is started to express a first indicating signal, and the output record of the first cell is 0; the promoter of the signal to be detected in the third line of the second cell is started to express the first indicating signal, and the output record of the second cell is 0; combining the first cell output record with the second cell output record, the system output record is 00.
Preferably, the method further comprises the following steps: when the signal to be detected and the first concomitant signal coexist, the signal to be detected promoter in the first circuit of the first cell is started to express the first concomitant signal promoter activating element and the first indicating signal, the first concomitant signal promoter in the second circuit of the first cell is started to express the first indicating signal inhibiting element and the second indicating signal, the first indicating signal inhibiting element can inhibit the expression of the first indicating signal, and the first cell only expresses the second indicating signal and outputs 1; the promoter of the signal to be detected in the third line of the second cell is started to express the first indicating signal, and the output of the second cell is 0; the system output record is 10 in combination with the first cell output record and the second cell output record.
Preferably, the method further comprises the following steps: when the signal to be detected and the second accompanying signal coexist, the promoter of the signal to be detected in the first circuit of the first cell is started to express a first indicating signal, and the output of the first cell is 0; the promoter of the signal to be detected in the third circuit of the second cell is started to express a second adjoint signal promoter activating element and a first indicating signal, the promoter of the second adjoint signal in the fourth circuit of the second cell is started to express a first indicating signal inhibiting element and a second indicating signal, the first indicating signal inhibiting element inhibits the expression of the first indicating signal, only the second indicating signal is expressed by the second cell, and the output is 1; the system output record is 01, combining the first cell output record with the second cell output record.
Preferably, the method further comprises the following steps: when the signal to be detected, the first accompanying signal and the second accompanying signal coexist, a promoter of the signal to be detected in a first circuit of the first cell is started to express a first accompanying signal promoter activating element and a first indicating signal, a promoter of the first accompanying signal in a second circuit of the first cell is started to express a first indicating signal inhibiting element and a second indicating signal, the first indicating signal inhibiting element inhibits the expression of the first indicating signal, only the second indicating signal is expressed in the first cell, and the output is 1; the promoter of the signal to be detected in the third circuit of the second cell is started to express a second adjoint signal promoter activating element and a first indicating signal, the promoter of the second adjoint signal in the fourth circuit of the second cell is started to express a first indicating signal inhibiting element and a second indicating signal, the first indicating signal inhibiting element inhibits the expression of the first indicating signal, only the second indicating signal is expressed by the second cell, and the output is 1; the system output record is 11 in combination with the first cell output record and the second cell output record.
Preferably, the method further comprises the following steps: when the signal to be detected and the concomitant signal do not exist, the first circuit of the first cell does not work, the promoter activating element of the first concomitant signal is not expressed, and the second circuit does not work; the third circuit of the second cell is inoperative, does not express the promoter activating element of the second concomitant signal, and the fourth circuit is inoperative; because all lines of the first cell and the second cell do not work, the indicating signal cannot be detected; or when the signal to be detected does not exist but the first concomitant signal exists, the first circuit of the first cell does not work, the promoter activating element of the first concomitant signal is not expressed, and the second circuit does not work; the third circuit of the second cell is inoperative, does not express the promoter activating element of the second concomitant signal, and the fourth circuit is inoperative; because all lines of the first cell and the second cell do not work, the indicating signal cannot be detected; or when the signal to be detected does not exist but a second concomitant signal exists, the first circuit of the first cell does not work, the promoter activating element of the first concomitant signal is not expressed, and the second circuit does not work; the third circuit of the second cell is inoperative, does not express the promoter activating element of the second concomitant signal, and the fourth circuit is inoperative; because all lines of the first cell and the second cell do not work, the indicating signal cannot be detected; or when the signal to be detected does not exist but a first accompanying signal and a second accompanying signal exist, the first circuit of the first cell does not work, the promoter activating element of the first accompanying signal is not expressed, and the second circuit does not work; the third circuit of the second cell is inoperative, does not express the promoter activating element of the second concomitant signal, and the fourth circuit is inoperative; because all lines of the first cell and the second cell are not working, the indication signal cannot be detected.
Preferably, the signal to be detected is arabinose (Ara), the first accompanying signal is isopropyl- β -D-thiogalactopyranoside (IPTG), the second accompanying signal is anhydrotetracycline (aTc), and the promoter of the signal to be detected adopts promoter P initiated by AraBADThe first adjoint signal promoter adopts IPTG-started promoter PlacThe second concomitant signal promoter adopts a promoter P started by aTctet。
Preferably, the gene line is calculated by activating a promoter and inhibiting an indicator signal, wherein an indicator signal inhibitor element is used for executing inhibition operation of the indicator signal, and the indicator signal inhibitor element adopts a CRISPR/dCas9 system or a CRISPR/ddCpf1 system.
Preferably, the promoter activation is achieved by using Cre or FLP recombinase to flip the promoter between the two inverted loxP/FRT sites.
Preferably, the promoter activation is achieved by using Cre or FLP recombinase to delete the terminator between two homologloxP/FRT sites after the promoter.
The bacterial cell calculation component with the indication signals of the priority under different accompanying signals provided by the invention has the advantages that different indications are made on the absence of the signals to be detected, the existence of only the signals to be detected, the simultaneous existence of the signals to be detected and the corresponding accompanying signals by designing a gene circuit and utilizing the activation of a promoter and the inhibition of the indication signals, and the final result is indicated by the output combination of multiple cells. The component can be used in biological computers, biological sensors and other related applications which need to judge the priority of a specific signal in different states. The method has the advantages of calculation accuracy, high efficiency and easiness in use.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings needed to be used in the description of the embodiments are briefly introduced as follows:
FIG. 1 is a diagram showing an exemplary design of a gene circuit according to the present invention;
FIG. 2 is a gene circuit diagram of the first embodiment of the present invention;
FIG. 3 is a gene circuit diagram of a third embodiment of the present invention;
FIG. 4 is a gene circuit diagram of a fifth embodiment of the present invention.
Detailed Description
The present application will now be described in further detail with reference to the accompanying drawings and examples.
In the following description, the terms "first" and "second" are used for descriptive purposes only and are not intended to indicate or imply relative importance. The following description provides embodiments of the disclosure, which may be combined or substituted for various embodiments, and this application is therefore intended to cover all possible combinations of the same and/or different embodiments described. Thus, if one embodiment includes feature A, B, C and another embodiment includes feature B, D, then this application should also be considered to include an embodiment that includes one or more of all other possible combinations of A, B, C, D, even though this embodiment may not be explicitly recited in text below.
The working principle of the bacterial cell calculation part of the indication signal under different accompanying signals is as follows:
assuming that the signal to be tested is S, the priority of S in different states (the priority is represented by a binary representation method commonly used in electronic components) is shown in the table I, S1 represents a first accompanying signal, and S2 represents a second accompanying signal.
Watch 1
Status of state Priority level
S 00
S，S1 10
S，S2 01
S，S1，S2 11
S-free signal No detection signal
The priority indicating part comprises two cells, namely a first cell and a second cell, wherein each cell has two expression states of a first indicating signal R1 and a second indicating signal R2, wherein the first indicating signal R1 represents '0' and the second indicating signal R2 represents '1'.
The gene circuit of the priority indication part is shown in FIG. 1. Wherein the S promoter is activated in the presence of an S signal, the S1 promoter is repressed by the S1 repression element requiring de-repression of the S1 promoter activating element and is therefore activated in the presence of both an S1 signal and the S1 promoter activating element, the S2 promoter is repressed by the S2 repression element requiring de-repression of the S2 promoter activating element and is therefore activated in the presence of both an S2 signal and the S2 promoter activating element.
When only S signal exists, the S promoter in the first circuit of the first cell is started to express R1, and the output of the first cell is 0; the S promoter in the third line of the second cell is started to express R1, and the output of the second cell is 0; the system output is 00.
When S signal and S1 signal coexist, S promoter is started in the first circuit of the first cell, S1 promoter activating element and R1 are expressed, S1 promoter is started in the second circuit of the first cell, R1 inhibiting element and R2 are expressed, R1 inhibiting element can inhibit R1 expression, only R2 is expressed in the first cell, and the output is 1; the S promoter in the third line of the second cell is started to express R1, and the output of the second cell is 0; the system output is 10.
When the S signal and the S2 signal coexist, the S promoter in the first line of the first cell is started to express R1, and the output of the first cell is 0; the S promoter is started in the third circuit of the second cell, the S2 promoter activating element and the R1 are expressed, the S2 promoter is started in the fourth circuit of the second cell, the R1 inhibiting element and the R2 are expressed, the R1 inhibiting element can inhibit the expression of R1, only the R2 is expressed by the second cell, and the output is 1; the system output is 01.
When S signals and S1 and S2 signals coexist, an S promoter is started in a first circuit of the first cell, an S1 promoter activating element and R1 are expressed, an S1 promoter is started in a second circuit of the first cell, an R1 inhibiting element and R2 are expressed, an R1 inhibiting element can inhibit the expression of R1, only R2 is expressed in the first cell, and the output is 1; the S promoter is started in the third circuit of the second cell, the S2 promoter activating element and the R1 are expressed, the S2 promoter is started in the fourth circuit of the second cell, the R1 inhibiting element and the R2 are expressed, the R1 inhibiting element can inhibit the expression of R1, only the R2 is expressed by the second cell, and the output is 1; the system output is 11.
When the S signal is absent, the first line is inactive and does not express the S1 promoter activating element regardless of the presence or absence of the concomitant signal (including four states of absence of the concomitant signal, presence of only the S1 signal, presence of only the S2 signal, presence of both the S1 signal and the S2 signal), and the second line is inactive regardless of the presence or absence of the S1 signal; for the second cell, the third line is inoperative, does not express the S2 promoter activating element, and the fourth line is inoperative regardless of the presence of the S2 signal; thus, all lines in the first cell and the second cell are not active and no indicator signal is detected.
The bacterial cell calculation component of the indicator signal with different priority under the accompanying signals can be expanded to three accompanying signals and more accompanying signal conditions, and the specific method is that each accompanying signal is added, one cell is added, and the promoter of the accompanying signal of the cell is correspondingly changed. If the third concomitant signal is increased, the third cell is increased, and the third cell replaces the first concomitant signal promoter with the third concomitant signal promoter as compared to the first cell.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following is a detailed description of the cell calculation unit and its components of the present invention, which indicate the priority of signals under different accompanying signals, by way of example, with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example one
The signal S to be detected, the first companion signal S1, the second companion signal S2, the first indicator signal R1, and the second indicator signal R2 are shown in the following table.
Signal S to be detected Ara (arabinose)
First companion signal S1 IPTG (isopropyl- β -D-thiogalactopyranoside)
Second companion signal S2 aTc (anhydrotetracycline)
First indicator signal R1 Green fluorescent protein GFP (i.e. green light represents 0)
Second indicator signal R2 Red fluorescent protein RFP (i.e. Red light 1)
The promoter is specifically as follows: the S promoter adopts Ara-initiated promoter PBADThe S1 promoter adopts IPTG-started promoter PlacS2 promoter the aTc-promoted promoter P was usedtet。
In the embodiment, the CRISPR/dCas9 system is used to realize the function of indicating signal inhibition, the dCas9 protein is a protein which loses the cleavage activity of Cas9, can not cleave DNA, but can be combined with DNA to prevent transcription, and sgRNA is used to guide the dCas9 protein to be combined with a corresponding DNA fragment, so that the transcription is prevented from occurring, and a gene element cannot be expressed.
This example utilizes a system of DNA site-directed recombinases to activate the S1 and S2 promoters, wherein the DNA site-directed recombinases can recognize specific DNA sites and mediate DNA recombination between the recognition sites, and the orientation of the recognition sites will determine the form of recombination, such as deletion or inversion. This example accomplishes the activation function by promoter inversion. The DNA site-directed recombinase used in this example is Cre recombinase, and the recombination site corresponding thereto is loxP site. The Cre recombinase can flip the fragment between the two inverted loxP sites.
The gene circuit diagram of the present example is shown in fig. 2, and specifically, the operation principle related to fig. 2 is as follows: when only Ara signal is present, P is in the first line of the first cellBADPromoter is started to express GFP, and the output of the first cell is 0; p in third line of second cellBADPromoter is started to express GFP, and the output of the second cell is 0; the system output is 00.
When Ara signal and IPTG signal coexist, P is in the first line of the first cellBADPromoter is started to express Cre recombinase and GFP, and Cre recombinase turns over two homologous lines in the second circuit of the first cellTo P between loxP siteslacPromoter, PlacThe orientation of the promoter is changed from reverse to forward, so that P is present in the second line of the first celllacPromoter is started to express sgRNA _ GFP and RFP, the sgRNA _ GFP guides dCas9 protein to inhibit transcription of GFP, only the RFP is expressed in a first cell, and the output is 1; p in third line of second cellBADPromoter is started to express GFP, and the output of the second cell is 0; the system output is 10.
When Ara signal and aTc signal coexist, P is in the first line of the first cellBADPromoter is started to express GFP, and the output of the first cell is 0; p in third line of second cellBADPromoter is started to express Cre recombinase and GFP, and Cre recombinase flips P between two homologus loxP sites in the fourth circuit of the second celltetPromoter, PtetThe orientation of the promoter is changed from reverse to forward, so that P is present in the fourth line of the second celltetPromoter is started to express sgRNA _ GFP and RFP, the sgRNA _ GFP guides dCas9 protein to inhibit transcription of GFP, and only the RFP is expressed in the second cell, and the output is 1; the system output is 01.
When Ara signal and IPTG, aTc signal coexist, P is in the first line of the first cellBADPromoter is started to express Cre recombinase and GFP, and Cre recombinase flips P between two homologus loxP sites in the second circuit of the first celllacPromoter, PlacThe orientation of the promoter is changed from reverse to forward, so that P is present in the second line of the first celllacPromoter is started to express sgRNA _ GFP and RFP, the sgRNA _ GFP guides dCas9 protein to inhibit transcription of GFP, only the RFP is expressed in a first cell, and the output is 1; p in third line of second cellBADPromoter is started to express Cre recombinase and GFP, and Cre recombinase flips P between two homologus loxP sites in the fourth circuit of the second celltetPromoter, PtetThe orientation of the promoter is changed from reverse to forward, so that P is present in the fourth line of the second celltetPromoter is started to express sgRNA _ GFP and RFP, the sgRNA _ GFP guides dCas9 protein to inhibit transcription of GFP, and only the RFP is expressed in the second cell, and the output is 1; the system output is 11.
When Ara signalIn the absence of the concomitant signal, regardless of the presence or absence of the concomitant signal (including four states of absence of the concomitant signal, presence of only the first concomitant signal IPTG, presence of only the second concomitant signal aTc, and presence of both the first concomitant signal IPTG and the second concomitant signal aTc), the first line does not operate and Cre recombinase is not expressed in the first cell, and P in the second linelacThe promoter is in a reverse state, and the second circuit does not work no matter whether IPTG signals exist or not; for the second cell, the third circuit is not operated, Cre recombinase is not expressed, and P in the fourth circuittetThe promoter is in the reverse state, and the fourth line does not work regardless of whether the aTc signal exists or not; thus, all lines in the first cell and the second cell are not active, no indicator signal is detected, and the system has no output.
According to the example shown in fig. 2, the sgRNA design is required by the CRISPR/dCas9 system, and should be selected or constructed to ensure Ptet、PBAD、PlacColi strain or other strains working with CRISPR/dCas 9. The gene circuit is constructed on the plasmid, and different plasmid circuits in each cell should select different resistance as the screening marker. Plasmid construction and transformation into host cells follow conventional molecular cloning procedures. The signal input adopts a conventional inducible expression method. The result detection can be performed by using equipment capable of detecting fluorescent protein, such as a fluorescent microscope, a flow cytometer and the like.
Example two
In this example, the CRISPR/dCas9 system of the first example is replaced by CRISPR/ddCpf1, ddCpf1 protein, like dCas9 protein, can not cut DNA, but can bind to DNA to prevent transcription, and sgRNA is used to guide ddCpf1 protein to bind to corresponding DNA fragment to prevent transcription from occurring, so that a genetic element cannot be expressed. An E.coli strain or other strains capable of ensuring the work of CRISPR/ddCpf1 should be selected or constructed, the design of sgRNA is only required according to the requirements of a CRISPR/ddCpf1 system, and the rest is the same as the embodiment I.
EXAMPLE III
In this example, the Cre recombinase and loxP sites of the first example were replaced with FLP recombinase and frt sites, as shown in FIG. 3, and the rest were the same as those of the first example. The FLP recombinase functions similarly to Cre recombinase, with frt sites being their counterparts, and can delete a fragment between two homo-frt sites and flip a fragment between two reverse frt sites.
Example four
In this example, the Cre recombinase and loxP sites of the second example were replaced with FLP recombinase and frt sites, and the rest were the same as in the first example.
EXAMPLE five
In this embodiment, the promoter is activated by inverting the promoter in the first embodiment, and the promoter is activated by deleting the terminator after the promoter, which is the same as the first embodiment, and the gene circuit of this embodiment is shown in fig. 4. Specifically, the working principle of the present example is as follows: when only Ara signal is present, P is in the first line of the first cellBADPromoter is started to express GFP, and the output of the first cell is 0; p in third line of second cellBADPromoter is started to express GFP, and the output of the second cell is 0; the system output is 00.
When Ara signal and IPTG signal coexist, P is in the first line of the first cellBADPromoter, expression of Cre recombinase and GFP, and deletion of P in the second line of the first cell by Cre recombinaselacTerminator after promoter, PlacThe promoter starts to express sgRNA _ GFP and RFP, the sgRNA _ GFP guides dCas9 protein to inhibit the transcription of GFP, the first cell only expresses RFP, and the output is 1; p in third line of second cellBADPromoter is started to express GFP, and the output of the second cell is 0; the system output is 10.
When Ara signal and aTc signal coexist, P is in the first line of the first cellBADPromoter is started to express GFP, and the output of the first cell is 0; p in third line of second cellBADPromoter, expression of Cre recombinase and GFP, and deletion of P in fourth line of second cell by Cre recombinasetetTerminator after promoter, PtetThe promoter starts to express sgRNA _ GFP and RFP, the sgRNA _ GFP guides dCas9 protein to inhibit transcription of GFP, and only the RFP is expressed in the second cell, and the output is 1; the system output is 01.
When Ara signal and IPTG, aP in the first line of the first cell in the presence of Tc signalBADPromoter, expression of Cre recombinase and GFP, and deletion of P in the second line of the first cell by Cre recombinaselacTerminator after promoter, PlacThe promoter starts to express sgRNA _ GFP and RFP, the sgRNA _ GFP guides dCas9 protein to inhibit the transcription of GFP, the first cell only expresses RFP, and the output is 1; p in third line of second cellBADPromoter, expression of Cre recombinase and GFP, and deletion of P in fourth line of second cell by Cre recombinasetetTerminator after promoter, PtetThe promoter starts to express sgRNA _ GFP and RFP, the sgRNA _ GFP guides dCas9 protein to inhibit transcription of GFP, and only the RFP is expressed in the second cell, and the output is 1; the system output is 11.
When the Ara signal is absent, the first line does not work and Cre recombinase is not expressed in the first cell, and the P in the second line does not work regardless of the presence or absence of the concomitant signals (including four states of absence of the concomitant signal, presence of only the first concomitant signal IPTG, presence of only the second concomitant signal aTc, and presence of both the first concomitant signal IPTG and the second concomitant signal aTc)lacA terminator exists behind the promoter, and the second line does not work regardless of existence of IPTG signals; for the second cell, the third circuit is not operated, Cre recombinase is not expressed, and P in the fourth circuittetA terminator is arranged behind the promoter, and the fourth line does not work regardless of whether an aTc signal exists or not; thus, all lines in the first cell and the second cell are not active, no indicator signal is detected, and the system has no output.
The invention provides a cell counting component for indicating the priority of signals under different accompanying signals. The component can be used in relative applications such as biological computers, biosensors and the like which need to judge the priority of a specific signal under different states, realize the priority indication of the same signal under different accompanied signal states by combining two or even a plurality of cells and output results, change the output results by activating a promoter and inhibiting an indication signal, and realize the calculation function. The method has the advantages of calculation accuracy, high efficiency and easiness in use.
In addition, in order to facilitate understanding and applying a cell calculation means that indicates the priority of a signal under different accompanying signals, proposed by the present disclosure, the following explanation examples are given for the method of use of the means. It should be noted that the scope of the present disclosure is not limited to the following description.
Specifically, first, the gene line is assembled based on the gene line map. The specific gene circuit construction part works as a connecting gene part, the parts in the gene circuit are functional DNA fragments, DNA connection adopts DNA ligase as a tool, the DNA ligase is a commercial product, and products of various companies have corresponding use instructions and can be operated according to the instructions.
Taking the NEB T4DNA ligase commonly used in the laboratory as an example, the ligation process is as follows:
① the linkage system is constructed as follows:
reagent Dosage (mu L)
T4DNA ligase buffer (10X) 2
Vector DNA 2
Insert DNA 2
T4DNA ligase 1
ddH2O 13
Total of 20
② reaction conditions
16 hours at 16 ℃.
③ result detection
And transforming the ligation product into a competent cell, culturing, and selecting a single colony for sequencing verification.
Further, the gene circuit is encapsulated into the cell. Specifically, cell encapsulation refers to transformation of the constructed gene circuit into a suitable cell. Transformation refers to the process of entering plasmid into cells by thermal stimulation or electrical stimulation, and is a mature technology in molecular biology, and the thermal stimulation is commonly used in laboratories, and generally comprises the following specific steps: taking a tube (100 mu L) of competent cells, adding the plasmid (or the ligation product), and standing on ice for 30 minutes; the tube was placed in a 42 ℃ water bath for 90 seconds and rapidly cooled in an ice bath for 2 minutes; adding 100 μ L LB culture medium, culturing at 37 deg.C for 30 min; and (5) plating the culture plate overnight, and selecting a single colony for sequencing verification.
The method for indicating the priority by using the invention comprises the following steps: and (3) the first cell and the second cell which are transformed into the gene circuit are divided into 1: 100 are respectively added into test tubes (marked as test tubes 1 and 2) containing LB liquid culture medium, and put into a shaking table for shaking culture for 6 to 8 hours until the OD600 is 0.6 to 0.8; adding samples to be detected into the test tubes 1 and 2 respectively, and placing the test tubes 1 and 2 into a shaking table for continuous shaking culture for 12-16 hours; the fluorescence of the tubes 1, 2 was observed under a fluorescence microscope and the results were recorded.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
The foregoing describes the general principles of the present disclosure in conjunction with specific embodiments, however, it is noted that the advantages, effects, etc. mentioned in the present disclosure are merely examples and are not limiting, and they should not be considered essential to the various embodiments of the present disclosure. Furthermore, the foregoing disclosure of specific details is for the purpose of illustration and description and is not intended to be limiting, since the disclosure is not intended to be limited to the specific details so described.
Also, as used herein, the use of "or" in a list of items beginning with "at least one" indicates a separate list, e.g., "A, B or at least one of C" means A or B or C, or AB or AC or BC, or ABC (i.e., A and B and C). Furthermore, the word "exemplary" does not mean that the described example is preferred or better than other examples.
The foregoing description has been presented for purposes of illustration and description. Furthermore, this description is not intended to limit embodiments of the disclosure to the form disclosed herein. While a number of example aspects and embodiments have been discussed above, those of skill in the art will recognize certain variations, modifications, alterations, additions and sub-combinations thereof.

Claims (10)

1. A bacterial cell calculation member indicating the priority of signals under different concomitant signals, characterized in that the bacterial cell calculation member is composed of two cells, a first cell for detecting a signal to be detected and a first concomitant signal and a second cell for detecting a signal to be detected and a second concomitant signal;
the gene circuit in the first cell comprises a first circuit and a second circuit;
the first circuit consists of a signal promoter to be detected, a first adjoint signal promoter activating element, a first indicating signal and a terminator in sequence;
the second circuit is sequentially composed of a first adjoint signal promoter, a first adjoint signal promoter suppression element, a first indication signal suppression element, a second indication signal and a terminator;
the gene circuit in the second cell comprises a third circuit and a fourth circuit;
the third circuit consists of a signal promoter to be detected, a second adjoint signal promoter activating element, a first indicating signal and a terminator in sequence;
the fourth line is sequentially composed of a second adjoint signal promoter, a second adjoint signal promoter suppression element, a first indication signal suppression element, a second indication signal and a terminator;
the promoter of the signal to be detected is started by the signal to be detected, the first accompanying signal promoter is started by the first accompanying signal, the second accompanying signal promoter is started by the second accompanying signal, the promoter inhibiting element can inhibit the promoter from working, the promoter activating element can relieve the inhibition of the promoter inhibiting element on the promoter, and the indicating signal inhibiting element can inhibit the expression of the indicating signal.
2. A bacterial cell calculation component for indicating signal priority under different concomitant signals as claimed in claim 1 further comprising: when only the signal to be detected exists, the promoter of the signal to be detected in the first circuit of the first cell is started to express a first indicating signal, and the output record of the first cell is 0;
the promoter of the signal to be detected in the third line of the second cell is started to express the first indicating signal, and the output record of the second cell is 0;
combining the first cell output record with the second cell output record, the system output record is 00.
3. A bacterial cell calculation component for indicating signal priority under different concomitant signals as claimed in claim 1 further comprising: when the signal to be detected and the first concomitant signal coexist, the signal to be detected promoter in the first circuit of the first cell is started to express the first concomitant signal promoter activating element and the first indicating signal, the first concomitant signal promoter in the second circuit of the first cell is started to express the first indicating signal inhibiting element and the second indicating signal, the first indicating signal inhibiting element can inhibit the expression of the first indicating signal, and the first cell only expresses the second indicating signal and outputs 1;
the promoter of the signal to be detected in the third line of the second cell is started to express the first indicating signal, and the output of the second cell is 0;
the system output record is 10 in combination with the first cell output record and the second cell output record.
4. A bacterial cell calculation component for indicating signal priority under different concomitant signals as claimed in claim 1 further comprising: when the signal to be detected and the second accompanying signal coexist, the promoter of the signal to be detected in the first circuit of the first cell is started to express a first indicating signal, and the output of the first cell is 0;
the promoter of the signal to be detected in the third circuit of the second cell is started to express a second adjoint signal promoter activating element and a first indicating signal, the promoter of the second adjoint signal in the fourth circuit of the second cell is started to express a first indicating signal inhibiting element and a second indicating signal, the first indicating signal inhibiting element inhibits the expression of the first indicating signal, only the second indicating signal is expressed by the second cell, and the output is 1;
the system output record is 01, combining the first cell output record with the second cell output record.
5. A bacterial cell calculation component for indicating signal priority under different concomitant signals as claimed in claim 1 further comprising: when the signal to be detected, the first accompanying signal and the second accompanying signal coexist, a promoter of the signal to be detected in a first circuit of the first cell is started to express a first accompanying signal promoter activating element and a first indicating signal, a promoter of the first accompanying signal in a second circuit of the first cell is started to express a first indicating signal inhibiting element and a second indicating signal, the first indicating signal inhibiting element inhibits the expression of the first indicating signal, only the second indicating signal is expressed in the first cell, and the output is 1;
the promoter of the signal to be detected in the third circuit of the second cell is started to express a second adjoint signal promoter activating element and a first indicating signal, the promoter of the second adjoint signal in the fourth circuit of the second cell is started to express a first indicating signal inhibiting element and a second indicating signal, the first indicating signal inhibiting element inhibits the expression of the first indicating signal, only the second indicating signal is expressed by the second cell, and the output is 1;
the system output record is 11 in combination with the first cell output record and the second cell output record.
6. A bacterial cell calculation component for indicating signal priority under different concomitant signals as claimed in claim 1 further comprising: when the signal to be detected and the concomitant signal do not exist, the first circuit of the first cell does not work, the promoter activating element of the first concomitant signal is not expressed, and the second circuit does not work; the third circuit of the second cell is inoperative, does not express the promoter activating element of the second concomitant signal, and the fourth circuit is inoperative; because all lines of the first cell and the second cell do not work, the indicating signal cannot be detected; or
When the signal to be detected does not exist but a first concomitant signal exists, the first circuit of the first cell does not work, the promoter activating element of the first concomitant signal is not expressed, and the second circuit does not work; the third circuit of the second cell is inoperative, does not express the promoter activating element of the second concomitant signal, and the fourth circuit is inoperative; because all lines of the first cell and the second cell do not work, the indicating signal cannot be detected; or
When the signal to be detected does not exist but a second concomitant signal exists, the first circuit of the first cell does not work, the promoter activating element of the first concomitant signal is not expressed, and the second circuit does not work; the third circuit of the second cell is inoperative, does not express the promoter activating element of the second concomitant signal, and the fourth circuit is inoperative; because all lines of the first cell and the second cell do not work, the indicating signal cannot be detected; or
When the signal to be detected does not exist but a first accompanying signal and a second accompanying signal exist, the first circuit of the first cell does not work, the promoter activating element of the first accompanying signal is not expressed, and the second circuit does not work; the third circuit of the second cell is inoperative, does not express the promoter activating element of the second concomitant signal, and the fourth circuit is inoperative; because all lines of the first cell and the second cell are not working, the indication signal cannot be detected.
7. The bacterial cell calculation component for indicating signal priority under different concomitant signals of any one of claims 2-6, wherein the signal to be tested is arabinose Ara, the first concomitant signal is isopropyl- β -D-thiogalactopyranoside IPTG, the second concomitant signal is anhydrotetracycline aTc, and the promoter of the signal to be tested adopts promoter P initiated by AraBADThe first adjoint signal promoter adopts IPTG-started promoter PlacThe second concomitant signal promoter adopts a promoter P started by aTctet。
8. The bacterial cell calculation component of indicator signal priority under different concomitant signals according to claim 1, wherein the gene circuit is calculated by activating a promoter and inhibiting the indicator signal, wherein an indicator signal inhibitor element for performing the inhibition operation of the indicator signal employs CRISPR/dCas9 system or CRISPR/ddCpf1 system.
9. The bacterial cell computation element of claim 8, wherein said promoter activation is achieved by using Cre or FLP recombinase to flip the promoter between two inverted loxP/FRT sites.
10. A bacterial cell computation element according to claim 8, wherein said promoter activation is achieved by Cre or FLP recombinase deletion of the terminator between the two homologus loxP/FRT sites.
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