CN110288076A - Indication signal is in the different bacterial cell calculating units with priority under signal - Google Patents
Indication signal is in the different bacterial cell calculating units with priority under signal Download PDFInfo
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Abstract
Present disclose provides a kind of indication signals in the different bacterial cell calculating units with priority under signal.The cell calculating unit is by the two kinds of cell compositions of the first cell and the second cell, wherein the first cell is for detecting signal to be detected and first with signal, and the second cell is for detecting signal to be detected and second with signal；First intracellular genetic circuits include first line and the second route, and the second intracellular genetic circuits include tertiary circuit and the 4th route.Cell calculating unit provided by the invention, by designing genetic circuits, different instructions are made to no measured signal, only measured signal, measured signal and corresponding exist simultaneously with signal using the activation of promoter and the inhibition of indication signal, pass through the output combination instruction final result of many cells.The component can be used for biocomputer, biosensor etc. and need to judge that a certain signal specific in the related application of priority, has accuracy, high efficiency and the ease for use calculated under different conditions.
Description
Technical field
This disclosure relates to synthetic biology technical field, in particular to a kind of indication signal different with signal
The bacterial cell calculating unit of lower priority.
Background technique
The building of cell calculating unit is one of the hot issue of current synthetic biology.Currently, successfully constructing
Cell with or the cells calculating unit such as all kinds of logic gates and bistable switch, oscillator, memory such as non-.
Priority is computer system when handling multiple calculating tasks, determines that each task receives the preferential of system resource
The parameter of grade.Cytocomputer is limited by intracellular computing resource, to make limited computing resource by most effective use,
The material circumstance for usually requiring more each signal, determines its priority, and the signal of preferential answering high priority.Therefore, right
For cytocomputer, the judgement of priority is very important.Cytocomputer works in complicated biological chemical environment,
In environment signal kinds variation very greatly, for the same signal, although adjoint signal different present in environment not with
It is directly had an effect, but priority when may calculate it has an impact.Such as, for lactose, in no glucose
In the presence of, be for Escherichia coli it is essential, when there are glucose, then become not essential, importance change very
Greatly, priority may also change accordingly, currently, cytocomputer domain variability is without judging signal different with priority under signals
Calculating unit, immediate technology are cell size comparator.But existing cell size comparator can only be to three kinds of different objects
Matter carries out size comparison, and cannot be compared to size of the same substance under different conditions, and then indicate its priority.
Therefore the above problem has changed into urgently problem to be solved.
Summary of the invention
In order to solve the problems in the prior art, the embodiment of the present disclosure provides a kind of indication signal different with signals
The bacterial cell calculating unit of lower priority can be used for biocomputer, biosensor etc. and need to judge a certain signal specific
Under different conditions in the related application of priority.Same signal is realized by two kinds even the output result combination of various kinds of cell
Priority indication under different adjoint signal conditions, passes through the activation of promoter and the inhibition of indication signal, changes output knot
Fruit realizes computing function.Accuracy, high efficiency and ease for use with calculating.
In a first aspect, present disclose provides a kind of indication signals in the different bacterial cell calculating with priority under signal
Component, the bacterial cell calculating unit is by the two kinds of cell compositions of the first cell and the second cell, and first cell is for examining
Signal to be detected and first is surveyed with signal, second cell is for detecting signal to be detected and second with signal；
Described first intracellular genetic circuits include first line and the second route；
The first line is successively by measured signal promoter, first with the sub- active element of signal enabling, the first instruction
Signal, terminator composition；
Second route is successively by first with signal enabling, first with the sub- straining element of signal enabling, first
Indication signal straining element, the second indication signal, terminator composition；
Described second intracellular genetic circuits include tertiary circuit and the 4th route；
The tertiary circuit is successively by measured signal promoter, second with the sub- active element of signal enabling, the first instruction
Signal, terminator composition；
4th route is successively by second with signal enabling, second with the sub- straining element of signal enabling, first
Indication signal straining element, the second indication signal, terminator composition；
Wherein, measured signal promoter is started by measured signal, and first is opened by first with signal with signal enabling
It is dynamic, second with signal enabling by second with signal enabling, promoter straining element can inhibit promoter to work, promoter
Active element can release inhibition of the promoter straining element to promoter, and indication signal straining element can inhibit the table of indication signal
It reaches.
Preferably, further includes: in the presence of the only described measured signal, measured signal described in the first cell first line
Promoter starting, expresses the first indication signal, and the output of the first cell is recorded as 0；Letter to be measured described in second cell tertiary circuit
The starting of number promoter, expresses the first indication signal, and the output of the second cell is recorded as 0；In conjunction with first cell output record with
The second cell output record, system output are recorded as 00.
Preferably, further includes: when the measured signal and first with signal it is common in the presence of, the first cell first line
Described in the starting of measured signal promoter, expression described first is with the sub- active element of signal enabling and the first indication signal, the
First starts with signal enabling in one the second route of cell, expresses the first indication signal straining element and the second instruction letter
Number, the first indication signal straining element will inhibit the expression of the first indication signal, and the first cell only has the expression of the second indication signal,
Output is 1；The starting of measured signal promoter described in second cell tertiary circuit, expresses the first indication signal, and the second cell is defeated
It is out 0；It is recorded in conjunction with the first cell output record and the output of the second cell, system output is recorded as 10.
Preferably, further includes: when the measured signal and second with signal it is common in the presence of, the first cell first line
Described in measured signal promoter starting, express the first indication signal, the first cell output be 0；In second cell tertiary circuit
The measured signal promoter starting, expression second is with the sub- active element of signal enabling and the first indication signal, the second cell
Second starts with signal enabling in 4th route, the first indication signal straining element of expression and the second indication signal, and first
Indication signal straining element will inhibit the expression of the first indication signal, and the second cell only has the expression of the second indication signal, exports and be
1；It is recorded in conjunction with the first cell output record and the output of the second cell, system output is recorded as 01.
Preferably, further includes: when the measured signal and first with signal, second with signal it is common in the presence of,
The starting of measured signal promoter described in one cell first line, expression first refer to the sub- active element of signal enabling and first
Show signal, first starts with signal enabling in first the second route of cell, expresses the first indication signal straining element and the
Two indication signals, the first indication signal straining element will inhibit the expression of the first indication signal, and the first cell only has the second instruction
Signal representation, exporting is 1；The starting of measured signal promoter described in second cell tertiary circuit, expression second are opened with signal
Mover active element and the first indication signal, second starts with signal enabling in the 4th route of the second cell, expression first
Indication signal straining element and the second indication signal, the first indication signal straining element will inhibit the expression of the first indication signal,
Second cell only has the expression of the second indication signal, and exporting is 1；It is recorded in conjunction with the first cell output record and the output of the second cell,
System output is recorded as 11.
Preferably, further includes: when the measured signal and in the absence of signal, the first line of the first cell not work
Make, does not express first with the sub- active element of signal enabling, the second line-down；The tertiary circuit of second cell does not work,
Second is not expressed with the sub- active element of signal enabling, the 4th line-down；Because the first cell and the second cell is all
Line-down can not detect indication signal；Or when the measured signal is not present, but there are first with signal, the
The first line of one cell does not work, and does not express first with the sub- active element of signal enabling, the second line-down；Second is thin
The tertiary circuit of born of the same parents does not work, and does not express second with the sub- active element of signal enabling, the 4th line-down；Because first is thin
All line-downs of born of the same parents and the second cell, can not detect indication signal；Or when the measured signal is not present, but exist
Second with signal when, the first line of the first cell does not work, do not express first with the sub- active element of signal enabling, second
Line-down；The tertiary circuit of second cell does not work, and does not express second with the sub- active element of signal enabling, the 4th route
It does not work；Because all line-downs of the first cell and the second cell, can not detect indication signal；Or when described to be measured
Signal is not present, but when there are first with signal and second with signal, the first line of the first cell does not work, and does not express
First with the sub- active element of signal enabling, the second line-down；The tertiary circuit of second cell does not work, and does not express second
With the sub- active element of signal enabling, the 4th line-down；Because of all line-downs of the first cell and the second cell,
It can not detect indication signal.
Preferably, measured signal be arabinose (Ara), first with signal be isopropyl-beta D-thio galactopyranosyl
Glucosides (IPTG), second with signal be dehydration tetracycline (aTc), the measured signal promoter use Ara starting starting
Sub- PBAD, described first uses the promoter P of IPTG starting with signal enablinglac, described second adopts with signal enabling
The promoter P started with aTctet。
Preferably, the genetic circuits are to realize and calculate by the activation of promoter and the inhibition of indication signal, wherein are referred to
Show that signal straining element uses CRISPR/ for executing the inhibition operation to indication signal, the indication signal straining element
DCas9 system or CRISPR/ddCpf1 system.
Preferably, the promoter activation is using between two reversed sites loxP/FRT of Cre or FLP recombinase overturning
Promoter is realized.
Preferably, the promoter activation deletes latter two loxP/FRT in the same direction of promoter using Cre or FLP recombinase
Terminator between site is realized.
A kind of indication signal provided by the invention passes through in the different bacterial cell calculating units with priority under signal
Genetic circuits are designed, using the activation of promoter and the inhibition of indication signal to no measured signal, only measured signal, letter to be measured
Number and corresponding existed simultaneously with signal make different instructions, pass through the output combination instruction final result of many cells.The component
It can be used for the related application that biocomputer, biosensor etc. need to judge a certain signal specific priority under different conditions
In.Accuracy, high efficiency and ease for use with calculating.
Detailed description of the invention
In order to illustrate more clearly of the technical solution of the embodiment of the present disclosure, below to needed in embodiment description
Attached drawing is briefly described:
Fig. 1 is the design example figure of genetic circuits of the present invention；
Fig. 2 is the genetic circuits figure of first embodiment of the invention；
Fig. 3 is the genetic circuits figure of third embodiment of the invention；
Fig. 4 is the genetic circuits figure of fifth embodiment of the invention.
Specific embodiment
The application is further discussed in detail with reference to the accompanying drawings and examples.
In following introductions, term " first ", " second " only for descriptive purposes, and should not be understood as instruction or dark
Show relative importance.Following introductions provide multiple embodiments of the disclosure, can replace or merge between different embodiments
Combination, therefore the application is it is also contemplated that all possible combinations comprising documented identical and/or different embodiments.Thus, such as
Fruit one embodiment include feature A, B, C, another embodiment include feature B, D, then the application also should be regarded as include containing
A, the every other possible combined embodiment of one or more of B, C, D, although the embodiment may be in the following contents
In have specific literature record.
The indication signal is in the different working principles with the bacterial cell calculating unit of priority under signal are as follows:
It is assumed that measured signal is S, S in the priority under different conditions, (commonly use using in electronic component by the expression of priority
Binary representation) as shown in Table 1, S1 represents first with signal, and S2 represents second with signal.
Table one
| State | Priority |
| S | 00 |
| S, S1 | 10 |
| S, S2 | 01 |
| S, S1, S2 | 11 |
| Without S signal | Without detection signal |
The priority indication component includes two kinds of cells of the first cell and the second cell, and every kind of cell has the first indication signal
Two kinds of expression status of R1 and the second indication signal R2, wherein the first indication signal R1 represents ' 0 ', and the second indication signal R2 is represented
‘1’。
The genetic circuits of the priority indication component are as shown in Figure 1.Wherein, S promoter starting when there are S signal, S1
Promoter is inhibited by S1 straining element, needs S1 promoter active element derepression, therefore exist concurrently with S1 signal and S1
Starting when promoter active element, S2 promoter is inhibited by S2 straining element, S2 promoter active element derepression is needed,
Starting when therefore existing concurrently with S2 signal and S2 promoter active element.
In the presence of only S signal, S promoter starts in the first cell first line, expresses R1, and the output of the first cell is
0；S promoter starts in second cell tertiary circuit, expresses R1, and the output of the second cell is 0；System output is 00.
In the presence of S signal and S1 signal are common, S promoter starts in the first cell first line, expresses S1 promoter
Active element and R1, S1 promoter starting in first the second route of cell, expresses R1 straining element and R2, R1 straining element will
Inhibit the expression of R1, the first cell only has R2 expression, and exporting is 1；S promoter starts in second cell tertiary circuit, expresses R1,
The output of second cell is 0；System output is 10.
In the presence of S signal and S2 signal are common, S promoter starts in the first cell first line, expresses R1, and first is thin
Born of the same parents' output is 0；S promoter starts in second cell tertiary circuit, expresses S2 promoter active element and R1, the second cell the
S2 promoter starts in four routes, expresses R1 straining element and R2, R1 straining element will inhibit the expression of R1, the second cell only has
R2 expression, exporting is 1；System output is 01.
In the presence of S signal and S1, S2 signal are common, S promoter starts in the first cell first line, expression S1 starting
Sub- active element and R1, S1 promoter starting in first the second route of cell, expresses R1 straining element and R2, R1 straining element will
Inhibit the expression of R1, the first cell only has R2 expression, and exporting is 1；S promoter starts in second cell tertiary circuit, expresses S2
Promoter active element and R1, S2 promoter starting in the 4th route of the second cell, expresses R1 straining element and R2, R1 inhibit
Element will inhibit the expression of R1, and the second cell only has R2 expression, and exporting is 1；System output is 11.
In the absence of S signal, in spite of exist with signal (including there is no with signal, there is only S1 signal,
There is only S2 signal, exist simultaneously four kinds of states of S1 signal and S2 signal), for the first cell, first line does not work, no
S1 promoter active element is expressed, in spite of there are S1 signal, the second route does not work；For the second cell, third
Line-down does not express S2 promoter active element, and in spite of there are S2 signal, the 4th route does not work；Therefore,
All routes in one cell and the second cell do not work, can't detect any indication signal.
The indication signal can be extended to three kinds with letter in the different bacterial cell calculating units with priority under signal
Number and it is more with signal(l)ing condition, specific method is, it is every to increase a kind of adjoint signal, then increase a kind of cell, corresponding change is somebody's turn to do
Adjoint signal enabling of cell.Such as increase third with signal, then increases third cell, compared to the first cell,
Three cell thirds are with signal enabling filial generation for first with signal enabling.
In order to make the objectives, technical solutions, and advantages of the present invention clearer, by the following examples, it and combines attached
Figure, to a kind of indication signal of the present invention different with the cell calculating unit of priority and the specific embodiment party of component under signal
Formula is further elaborated.It should be appreciated that described herein, specific examples are only used to explain the present invention, is not used to
Limit the present invention.
Embodiment one
Signal S to be detected, first are with signal S1, second with signal S2, the first indication signal R1, the second indication signal
R2 is as shown in the table.
| Signal S to be detected | Ara (arabinose) |
| First with signal S1 | IPTG (isopropyl-beta D-thio galactopyranoside) |
| Second with signal S2 | ATc (dehydration tetracycline) |
| First indication signal R1 | Green fluorescent protein GFP (i.e. green light indicates 0) |
| Second indication signal R2 | Red fluorescent protein RFP (i.e. feux rouges indicates 1) |
Promoter specifically: S promoter uses the promoter P of Ara startingBAD, S1 promoter using IPTG starting open
Mover Plac, S2 promoter use aTc starting promoter Ptet。
The present embodiment realizes that indication signal inhibits function using CRISPR/dCas9 system, and dCas9 albumen is that one kind loses
The albumen of Cas9 cleavage activity, can not cutting DNA, but may be incorporated on DNA prevent transcription generation, guided using sgRNA
DCas9 albumen is in conjunction with corresponding DNA fragmentation, to prevent transcription that Genetic elements is made to be beyond expression.
The present embodiment realizes that S1, S2 promoter activate function, DNA fixed point recombination using DNA fixed point recombinase service system
Enzyme can identify the specific site DNA and the DNA between recognition site is mediated to recombinate, and the direction of recognition site will determine the shape of recombination
Formula is such as deleted or is overturn.The present embodiment is overturn by promoter realizes activation function.The DNA that the present embodiment uses pinpoints recombination
Enzyme is Cre recombinase, and corresponding recombination site is the site loxP.Cre recombinase can be overturn between two reversed sites loxP
Segment.
The genetic circuits figure of the present embodiment is as shown in Fig. 2, specifically, working principle involved in Fig. 2 are as follows: as only Ara
In the presence of signal, P in the first cell first lineBADPromoter starting, expresses GFP, and the output of the first cell is 0；Second cell
P in three routesBADPromoter starting, expresses GFP, and the output of the second cell is 0；System output is 00.
In the presence of Ara signal and IPTG signal are common, P in the first cell first lineBADPromoter starting, expresses Cre
Recombinase and GFP, Cre recombinase overturn the P in first the second route of cell between two sites loxP in the same directionlacPromoter,
PlacThe direction of promoter is from reversely becoming positive, therefore, P in first the second route of celllacPromoter starting, expresses sgRNA_
GFP and RFP, sgRNA_GFP guide the transcription of dCas9 albumen inhibition GFP, and the first cell only has RFP expression, and exporting is 1；The
P in two cell tertiary circuitsBADPromoter starting, expresses GFP, and the output of the second cell is 0；System output is 10.
In the presence of Ara signal and aTc signal are common, P in the first cell first lineBADPromoter starting, expresses GFP,
The output of first cell is 0；P in second cell tertiary circuitBADPromoter starting, expresses Cre recombinase and GFP, Cre recombinase
Overturn the P in the 4th route of the second cell between two sites loxP in the same directiontetPromoter, PtetThe direction of promoter is by reversed
Become positive, therefore, P in the 4th route of the second celltetPromoter starting, expresses sgRNA_GFP and RFP, and sgRNA_GFP draws
The transcription that dCas9 albumen inhibits GFP is led, the second cell only has RFP expression, and exporting is 1；System output is 01.
In the presence of Ara signal and IPTG, aTc signal are common, P in the first cell first lineBADPromoter starting, table
Up to Cre recombinase and GFP, Cre recombinase overturns the P in first the second route of cell between two sites loxP in the same directionlacStarting
Son, PlacThe direction of promoter is from reversely becoming positive, therefore, P in first the second route of celllacPromoter starting, expression
SgRNA_GFP and RFP, sgRNA_GFP guide the transcription of dCas9 albumen inhibition GFP, and the first cell only has RFP expression, export
It is 1；P in second cell tertiary circuitBADPromoter starting, expresses Cre recombinase and GFP, Cre recombinase overturn the second cell
P in 4th route between two sites loxP in the same directiontetPromoter, PtetThe direction of promoter is from reversely becoming positive, therefore,
P in the 4th route of second celltetPromoter starting, expresses sgRNA_GFP and RFP, and sgRNA_GFP guides dCas9 albumen to inhibit
The transcription of GFP, the second cell only have RFP expression, and exporting is 1；System output is 11.
In the absence of Ara signal, in spite of exist with signal (including there is no with signal, there is only first
With signal IPTG, there is only second with signal aTc, exists simultaneously first with signal IPTG and second with signal aTc
Four kinds of states), for the first cell, first line does not work, and does not express Cre recombinase, the P in the second routelacPromoter
In reverse state, in spite of there are IPTG signal, the second route does not work；For the second cell, tertiary circuit is not
Work, does not express Cre recombinase, the P in the 4th routetetPromoter is in reverse state, in spite of there are aTc signal,
4th route does not work；Therefore, all routes in the first cell and the second cell do not work, can't detect any instruction
Signal, system is without output.
In example according to Fig.2, sgRNA is designed according to CRISPR/dCas9 system requirements, Ying Xuanyong or structure
It builds and can ensure that Ptet、PBAD、Plac, CRISPR/dCas9 work E.coli bacterial strain or other bacterial strains.Genetic circuits are implemented in plasmid
On, each intracellular different plasmid routes should select different resistances to indicate as screening.It plasmid construction and is transformed into host cell and abides by
Follow common molecular clone operations method.Signal input is using conventional derivational expression method.The optional fluorescence microscope of as a result detection,
The equipment of the detectable fluorescin such as flow cytometer.
Embodiment two
The present embodiment replaces the CRISPR/dCas9 system CRISPR/ddCpf1 of embodiment one, ddCpf1 albumen with
DCas9 albumen is similar, can not cutting DNA, but may be incorporated on DNA prevent transcription generation, utilize sgRNA guide ddCpf1 egg
It is white in conjunction with corresponding DNA fragmentation, thus prevent transcription so that Genetic elements is beyond expression.Should select or construct can ensure that
The E.coli bacterial strain or other bacterial strains of CRISPR/ddCpf1 work, sgRNA design are according to CRISPR/ddCpf1 system requirements
Can, other are the same as example 1.
Embodiment three
The present embodiment replaces the Cre recombinase of embodiment one and the site loxP with FLP recombinase and the site frt, such as schemes
3, other are the same as example 1.FLP recombinase is functionally similar to Cre recombinase, and the site frt is its corresponding site,
FLP recombinase can delete the segment between two sites frt in the same direction, can also overturn the segment between two reversed sites frt.
Example IV
The present embodiment replaces the Cre recombinase of embodiment two and the site loxP with FLP recombinase and the site frt, other
It is the same as example 1.
Embodiment five
The present embodiment activates the overturning promoter of embodiment one promoter to be changed to the termination after deletion promoter
Son activates promoter, other are the same as example 1, the genetic circuits of the present embodiment are as shown in Figure 4.Specifically, this example
Working principle are as follows: in the presence of only Ara signal, P in the first cell first lineBADPromoter starting, express GFP, first
Cell output is 0；P in second cell tertiary circuitBADPromoter starting, expresses GFP, and the output of the second cell is 0；System output
It is 00.
In the presence of Ara signal and IPTG signal are common, P in the first cell first lineBADPromoter starting, expresses Cre
Recombinase and GFP, Cre recombinase delete P in first the second route of celllacTerminator after promoter, PlacPromoter starting
SgRNA_GFP and RFP is expressed, sgRNA_GFP guides the transcription of dCas9 albumen inhibition GFP, and the first cell only has RFP expression,
Output is 1；P in second cell tertiary circuitBADPromoter starting, expresses GFP, and the output of the second cell is 0；System output is 10.
In the presence of Ara signal and aTc signal are common, P in the first cell first lineBADPromoter starting, expresses GFP,
The output of first cell is 0；P in second cell tertiary circuitBADPromoter starting, expresses Cre recombinase and GFP, Cre recombinase
Delete P in the 4th route of the second celltetTerminator after promoter, PtetPromoter starting expression sgRNA_GFP and RFP,
SgRNA_GFP guides the transcription of dCas9 albumen inhibition GFP, and the second cell only has RFP expression, and exporting is 1；System exports
01。
In the presence of Ara signal and IPTG, aTc signal are common, P in the first cell first lineBADPromoter starting, table
Up to Cre recombinase and GFP, Cre recombinase deletes P in first the second route of celllacTerminator after promoter, PlacStarting
Son starting expression sgRNA_GFP and RFP, sgRNA_GFP guide the transcription of dCas9 albumen inhibition GFP, and the first cell only has RFP
Expression, exporting is 1；P in second cell tertiary circuitBADPromoter starting, expresses Cre recombinase and GFP, Cre recombinase are deleted
Except P in the 4th route of the second celltetTerminator after promoter, PtetPromoter starting expression sgRNA_GFP and RFP,
SgRNA_GFP guides the transcription of dCas9 albumen inhibition GFP, and the second cell only has RFP expression, and exporting is 1；System output is 11.
In the absence of Ara signal, in spite of exist with signal (including there is no with signal, there is only first
With signal IPTG, there is only second with signal aTc, exists simultaneously first with signal IPTG and second with signal aTc
Four kinds of states), for the first cell, first line does not work, and does not express Cre recombinase, the P in the second routelacPromoter
There is terminator below, in spite of there are IPTG signal, the second route does not work；For the second cell, tertiary circuit
It does not work, does not express Cre recombinase, the P in the 4th routetetThere are terminators behind promoter, in spite of there are aTc letters
Number, the 4th route does not work；Therefore, all routes in the first cell and the second cell do not work, can't detect any
Indication signal, system is without output.
The present invention provides a kind of indication signals in the different cell calculating units with priority under signal.The component can
Need to judge the related application of a certain signal specific priority under different conditions for biocomputer, biosensor etc.
In, priority of the same signal under Bu Tong with signal condition is realized by two kinds of even various kinds of cell and output result combinations
Instruction changes output as a result, realizing computing function by the activation of promoter and the inhibition of indication signal.Essence with calculating
Parasexuality, high efficiency and ease for use.
In addition, a kind of indication signal proposed in order to facilitate understanding from the application disclosure priority under different adjoint signals
Cell calculating unit, the application method of component is carried out following to illustrate example.It should be noted that disclosure protection scope is not
It is limited to following explanation.
Specifically, firstly, assembling genetic circuits according to genetic circuits figure.The work of specific genetic circuits building part is
For linker because of component, the component in genetic circuits is functional DNA fragmentation, and DNA connection uses DNA ligase as tool,
DNA ligase is a kind of commercially produced product, and each Products have corresponding operation instructions, is operated to specifications.
By taking the common NEB T4DNA ligase in laboratory as an example, connection procedure are as follows:
1. it is as follows to construct linked system:
| Reagent | Dosage (μ L) |
| T4DNA ligase buffer solution (10 ×) | 2 |
| Carrier DNA | 2 |
| It is inserted into DNA | 2 |
| T4DNA ligase | 1 |
| ddH2O | 13 |
| It amounts to | 20 |
2. reaction condition
16 DEG C 16 hours.
3. result detects
Connection product is transformed into competent cell, cultivates, chooses single colonie sequence verification.
Further, genetic circuits are encapsulated into the cell.Specifically, cell encapsulation refers to the genetic circuits that will be built
It is transformed into suitable intracellular.Conversion refers to makes plasmid enter cell by thermostimulation or electro photoluminescence, is in molecular biology
A kind of mature technology commonly uses thermostimulation mode in laboratory, and general specific step is as follows: take competent cell one to manage (100 μ L),
It is added plasmid (or connection product), places 30 minutes on ice；Pipe is placed in 42 DEG C of water-baths 90 seconds, is cooled down rapidly in ice bath
2 minutes；100 μ L LB culture mediums are added, 37 DEG C are cultivated 30 minutes；Single colonie sequence verification is chosen in plated overnight culture.
Using the present invention instruction priority method and step it is as follows are as follows: by be transformed into genetic circuits the first cell, second
Cell is separately added into the test tube containing LB liquid medium by 1:100 (labeled as test tube 1,2), is put into shaken cultivation 6- in shaking table
8 hours to OD600 be 0.6-0.8 when；It is separately added into sample to be tested to test tube 1,2, test tube 1,2 is put into shaking table relaying persistent oscillation
Culture 12-16 hours；The fluorescence that test tube 1,2 is observed under fluorescence microscope, records result.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
The basic principle of the disclosure is described in conjunction with specific embodiments above, however, it is desirable to, it is noted that in the disclosure
The advantages of referring to, advantage, effect etc. are only exemplary rather than limitation, must not believe that these advantages, advantage, effect etc. are the disclosure
Each embodiment is prerequisite.In addition, detail disclosed above is merely to exemplary effect and the work being easy to understand
With, rather than limit, it is that must be realized using above-mentioned concrete details that above-mentioned details, which is not intended to limit the disclosure,.
In addition, as used herein, the "or" instruction separation used in the enumerating of the item started with "at least one"
Enumerate, for example, " at least one of A, B or C " enumerate mean A or B or C or AB or AC or BC or ABC (i.e. A and B and
C).In addition, wording " exemplary " does not mean that the example of description is preferred or more preferable than other examples.
Above description is had been presented for for purposes of illustration and description.In addition, this description is not intended to the reality of the disclosure
It applies example and is restricted to form disclosed herein.Although already discussed above multiple exemplary aspects and embodiment, this field skill
Its certain modifications, modification, change, addition and sub-portfolio will be recognized in art personnel.
Claims (10)
1. a kind of indication signal is in the different bacterial cell calculating units with priority under signal, which is characterized in that described thin
Bacterium cell calculating unit is by the two kinds of cell compositions of the first cell and the second cell, and first cell is for detecting signal to be detected
With first with signal, second cell is for detecting signal to be detected and second with signal；
Described first intracellular genetic circuits include first line and the second route；
The first line successively by measured signal promoter, first with the sub- active element of signal enabling, the first indication signal,
Terminator composition；
Second route is successively indicated with signal enabling, first with the sub- straining element of signal enabling, first by first
Signal straining element, the second indication signal, terminator composition；
Described second intracellular genetic circuits include tertiary circuit and the 4th route；
The tertiary circuit successively by measured signal promoter, second with the sub- active element of signal enabling, the first indication signal,
Terminator composition；
4th route is successively indicated with signal enabling, second with the sub- straining element of signal enabling, first by second
Signal straining element, the second indication signal, terminator composition；
Wherein, measured signal promoter is started by measured signal, first with signal enabling by first with signal enabling,
Two with signal enabling by second with signal enabling, promoter straining element can inhibit promoter to work, promoter activation
Element can release inhibition of the promoter straining element to promoter, and indication signal straining element can inhibit the expression of indication signal.
2. indication signal according to claim 1 is in the different cell calculating units with priority under signal, feature
It is, further includes: in the presence of the only described measured signal, measured signal promoter described in the first cell first line is opened
It is dynamic, the first indication signal is expressed, the output of the first cell is recorded as 0；
The first indication signal, the second cell output note are expressed in the starting of measured signal promoter described in second cell tertiary circuit
Record is 0；
It is recorded in conjunction with first cell output record and second cell output, system output is recorded as 00.
3. indication signal according to claim 1 is in the different cell calculating units with priority under signal, feature
Be, further includes: when the measured signal and first with signal it is common in the presence of, it is to be measured described in the first cell first line
The starting of signal enabling, expression described first is with the sub- active element of signal enabling and the first indication signal, the first cell second
First starts with signal enabling in route, expresses the first indication signal straining element and the second indication signal, the first instruction
Signal straining element will inhibit the expression of the first indication signal, and the first cell only has the expression of the second indication signal, and exporting is 1；
The starting of measured signal promoter described in second cell tertiary circuit, expresses the first indication signal, and the output of the second cell is
0；
It is recorded in conjunction with the first cell output record and the output of the second cell, system output is recorded as 10.
4. indication signal according to claim 1 is in the different cell calculating units with priority under signal, feature
Be, further includes: when the measured signal and second with signal it is common in the presence of, it is to be measured described in the first cell first line
The starting of signal enabling, expresses the first indication signal, and the output of the first cell is 0；
The starting of measured signal promoter described in second cell tertiary circuit, expression second with the sub- active element of signal enabling and
First indication signal, second starts with signal enabling in the 4th route of the second cell, and the first indication signal of expression inhibits member
Part and the second indication signal, the first indication signal straining element will inhibit the expression of the first indication signal, and the second cell only has the
The expression of two indication signals, exporting is 1；
It is recorded in conjunction with the first cell output record and the output of the second cell, system output is recorded as 01.
5. indication signal according to claim 1 is in the different cell calculating units with priority under signal, feature
Be, further includes: when the measured signal and first with signal, second with signal it is common in the presence of, the first cell first
The starting of measured signal promoter described in route, expression first is with the sub- active element of signal enabling and the first indication signal, and the
First starts with signal enabling in one cell and the second route, expresses the first indication signal straining element and the second instruction letter
Number, the first indication signal straining element will inhibit the expression of the first indication signal, and the first cell only has the expression of the second indication signal,
Output is 1；
The starting of measured signal promoter described in second cell tertiary circuit, expression second with the sub- active element of signal enabling and
First indication signal, second starts with signal enabling in the 4th route of the second cell, and the first indication signal of expression inhibits member
Part and the second indication signal, the first indication signal straining element will inhibit the expression of the first indication signal, and the second cell only has the
The expression of two indication signals, exporting is 1；
It is recorded in conjunction with the first cell output record and the output of the second cell, system output is recorded as 11.
6. indication signal according to claim 1 is in the different cell calculating units with priority under signal, feature
It is, further includes: when the measured signal and in the absence of signal, the first line of the first cell does not work, and does not express
First with the sub- active element of signal enabling, the second line-down；The tertiary circuit of second cell does not work, and does not express second
With the sub- active element of signal enabling, the 4th line-down；Because of all line-downs of the first cell and the second cell,
It can not detect indication signal；Or
When the measured signal is not present, but there are first with signal, the first line of the first cell does not work, and does not express
First with the sub- active element of signal enabling, the second line-down；The tertiary circuit of second cell does not work, and does not express second
With the sub- active element of signal enabling, the 4th line-down；Because of all line-downs of the first cell and the second cell,
It can not detect indication signal；Or
When the measured signal is not present, but there are second with signal, the first line of the first cell does not work, and does not express
First with the sub- active element of signal enabling, the second line-down；The tertiary circuit of second cell does not work, and does not express second
With the sub- active element of signal enabling, the 4th line-down；Because of all line-downs of the first cell and the second cell,
It can not detect indication signal；Or
When the measured signal is not present, but when there are first with signal and second with signal, the First Line of the first cell
Road does not work, and does not express first with the sub- active element of signal enabling, the second line-down；The tertiary circuit of second cell is not
Work, does not express second with the sub- active element of signal enabling, the 4th line-down；Because the first cell and the second cell
All line-downs can not detect indication signal.
7. according to the described in any item indication signals of claim 2-6 in the different cell calculation parts with priority under signal
Part, which is characterized in that measured signal be arabinose (Ara), first with signal be isopropyl-beta D-thio galactopyranose
Glycosides (IPTG), second with signal be dehydration tetracycline (aTc), the measured signal promoter use Ara starting promoter
PBAD, described first uses the promoter P of IPTG starting with signal enablinglac, described second uses with signal enabling
The promoter P of aTc startingtet。
8. indication signal according to claim 1 is in the different cell calculating units with priority under signal, feature
It is, the genetic circuits are to realize and calculate by the activation of promoter and the inhibition of indication signal, wherein indication signal inhibits
Element be used for executes to indication signal inhibition operation, the indication signal straining element use CRISPR/dCas9 system or
CRISPR/ddCpf1 system.
9. indication signal according to claim 8 is in the different cell calculating units with priority under signal, feature
It is, the promoter activation overturns the promoter between two reversed sites loxP/FRT using Cre or FLP recombinase and realizes.
10. indication signal according to claim 8 is in the different cell calculating units with priority under signal, feature
It is, the termination between latter two site loxP/FRT in the same direction of promoter is deleted in the promoter activation using Cre or FLP recombinase
Son is realized.
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