CN109784464A - A kind of bacterial cell sequence identifier and cytocomputer - Google Patents
A kind of bacterial cell sequence identifier and cytocomputer Download PDFInfo
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Abstract
The invention discloses a kind of bacterial cell sequence identifier and cytocomputers, belong to cytocomputer technical field, solve the technical issues of prior art can only indicate a kind of input sequence.Bacterial cell sequence identifier is by the two kinds of cell compositions of the first cell and the second cell, wherein the sequence of the first cell the first signal and the second signal for identification, the sequence of the second cell third signal and the first, second signal for identification；The genetic circuit of first cell includes the first circuit and second circuit；The genetic circuit of second cell includes tertiary circuit, the 4th circuit and the 5th circuit.Bacterial cell sequence identifier provided by the invention, by destroying circuit or element, it is allowed to no longer work from now on, carry out tracer signal input sequence, and realized by the output combination of two kinds of cells to the instruction of three kinds of six kinds of signal different input sequences, it can be used for biocomputer, biosensor etc. and need to determine in the related application of input sequence.
Description
Technical field
The present invention relates to cytocomputer technical field more particularly to a kind of bacterial cell sequence identifiers and cytometer
Calculation machine.
Background technique
The building of cell calculating unit is current one of hot issue.Currently, successfully constructed cell with or
The cells calculating unit such as all kinds of logic gates such as non-and bistable switch, memory, counter.
Sequence identifier is a kind of important calculating unit, and there has been no the relevant technologies sides of cell sequence identifier at present
Case.Immediate technology is that Collins study group in 2009 is constructed in bacterial cell using DNA fixed point recombinase Cre and FLP
The unicellular counter of multi signal (document source: A.E.Friedland, T.K.Lu, X.Wang, D.Shi, G.Church, and
J.J.Collins,"Synthetic
gene networks that count,"Science,vol.324,no.5931,pp.1199-1202,
May.2009).The counter is 3 counters, can be counted to the specific input sequence of 3 kinds of signals.There are three types of such as
Signal aTc (dehydration tetracycline), Ara (arabinose), IPTG (isopropyl-beta D-thio galactopyranoside), only exist
Under this input sequence of aTc-Ara-IPTG, the result mark (green fluorescence) of the counter could be generated.Merely entering one kind
In the case that signal, two kinds of signals are inputted in other sequences by different order input and three kinds of signals, as a result mark cannot
It generates.
Fig. 1 is the genetic circuit and working principle diagram of the unicellular counter of multi signal of Collins study group building, work
Make process are as follows: firstly, input aTc, starts PLtet0-1Promoter generates FlpeRecombinase, FlpeRecombinase overturns FRTfBetween site
Segment so that PBADPromoter from reversely becoming positive, meanwhile, FlpeIt is reversed to recombinate enzyme gene, does not regenerate FlpeRecombinase；
Secondly, input Ara, starts PBADPromoter, generates cre recombinase, and cre recombinase overturns loxPfSegment between site, so that
PA1lacOPromoter from reversely becoming positive, meanwhile, it is reversed that cre recombinates enzyme gene, does not regenerate cre recombinase；Finally, input
IPTG starts PA1lacOPromoter generates green fluorescent protein gfp, issues green fluorescence.
From figure 1 it appears that the multi signal cell counter utilizes the rollover characteristics of recombinase, by promoter and recombination
Enzyme is cascaded in certain sequence, indicates to realize a certain specific input sequence of three kinds of signals.However,
The shortcomings that multi signal unicellular counter of Collins study group building is can only to indicate a kind of input sequence, can not indicate it
His input sequence, greatly limits its application range.
Summary of the invention
The purpose of the present invention is in bacterial cell, construct a kind of to carry out all different input sequences of three kinds of signals
The sequence identifier of instruction.
To achieve the goals above, the invention provides the following technical scheme:
A kind of bacterial cell sequence identifier, by the two kinds of cell compositions of the first cell and the second cell, wherein first is thin
The sequence of born of the same parents' the first signal and the second signal for identification, the second cell third signal and the first, second signal for identification
Sequentially；
The genetic circuit of first cell includes the first circuit and second circuit, as shown in Figures 2 and 3；Wherein,
First circuit is successively destroyed element, fisrt feature signal and terminator and is formed by the first promoter, second circuit；
Second circuit is successively made of the second promoter, the first circuit damage element, second feature signal and terminator；
The genetic circuit of second cell includes tertiary circuit, the 4th circuit and the 5th circuit；Wherein,
Tertiary circuit is successively made of constitutive promoter, fisrt feature signal, second feature signal and terminator；
4th circuit successively destroys element, terminator, the second promoter, second feature by the first promoter, fisrt feature
Destroy element and terminator composition；
5th circuit is successively made of third promoter, the 4th circuit damage element and terminator；
Wherein, the first promoter is started by the first signal enabling, the second promoter by second signal, and third promoter is by
Three signal enablings, circuit damage element can destroy the work of entire circuit, and feature destroys element only destructive characteristics signal
Work.
Above-mentioned bacterial cell sequence identifier, the method for identification signal sequence are as follows:
(1) when original state,
First the first circuit of cell, second circuit do not work, non-output signal；
Only tertiary circuit works second cell, exports fisrt feature signal and second feature signal；
(2) when signal input sequence is 1-2-3,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, output first
Characteristic signal；After inputting second signal, since second circuit is destroyed, second feature signal is no longer exported；Input third signal
Afterwards, unchanged；
After second cell inputs the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal is not
It exports again；After inputting second signal, the second promoter starts second feature and destroys element, and second feature signal no longer exports, defeated
After entering third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
(3) when signal input sequence is 1-3-2,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, output first
Characteristic signal；It is unchanged after inputting third signal；After inputting second signal, since second circuit is destroyed, second is no longer exported
Characteristic signal；
After second cell inputs the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal is not
It exports again；After inputting third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input second
After signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature signal；
(4) when signal input sequence is 2-1-3,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, output second
Characteristic signal；After inputting the first signal, since the first circuit is destroyed, fisrt feature signal is no longer exported；Input third signal
Afterwards, unchanged；
After second cell inputs second signal, the second promoter starts second feature and destroys element, and second feature signal is not
It exports again；After inputting the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal no longer exports；It is defeated
After entering third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
(5) when signal input sequence is 2-3-1,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, output second
Characteristic signal；It is unchanged after inputting third signal；After inputting the first signal, since the first circuit is destroyed, first is no longer exported
Characteristic signal；
After second cell inputs second signal, the second promoter starts second feature and destroys element, and second feature signal is not
It exports again；After inputting third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input first
After signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature signal；
(6) when signal input sequence is 3-1-2,
It is unchanged after first cell inputs third signal；After inputting the first signal, the first promoter starts the first circuit,
Second circuit is destroyed, and exports fisrt feature signal；After inputting second signal, since second circuit is destroyed, no longer output the
Two characteristic signals；
After second cell inputs third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
After inputting the first signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature letter
Number；After inputting second signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, and it is special still to export second
Reference number；
(7) when signal input sequence is 3-2-1,
It is unchanged after first cell inputs third signal；After inputting second signal, the second promoter starts second circuit,
First circuit is destroyed, and exports second feature signal；After inputting the first signal, since the first circuit is destroyed, no longer output the
One characteristic signal；
After second cell inputs third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
After inputting second signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature letter
Number；After inputting the first signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, and it is special still to export first
Reference number.
The results are shown in Table 1 for the output of the first cell and the second cell under various signal input sequences.
First cell, the second cell export result under the various signal input sequences of table 1
Above-mentioned bacterial cell sequence identifier, the 4th circuit, which can be replaced, successively to be broken by the first promoter, second feature
Bad element, terminator, the second promoter, fisrt feature destroy element and terminator composition, as shown in Figure 4.
The replaced bacterial cell sequence identifier of the 4th circuit of second cell, the method for identification signal sequence are as follows:
(1) when original state,
First the first circuit of cell, second circuit do not work, non-output signal；
Only tertiary circuit works second cell, exports fisrt feature signal and second feature signal；
(2) when signal input sequence is 1-2-3,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, output first
Characteristic signal；After inputting second signal, since second circuit is destroyed, second feature signal is no longer exported；Input third signal
Afterwards, unchanged；
After second cell inputs the first signal, the first promoter starts second feature and destroys element, and second feature signal is not
It exports again；After inputting second signal, the second promoter starts fisrt feature and destroys element, and fisrt feature signal no longer exports, defeated
After entering third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
(3) when signal input sequence is 1-3-2,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, output first
Characteristic signal；It is unchanged after inputting third signal；After inputting second signal, since second circuit is destroyed, second is no longer exported
Characteristic signal；
After second cell inputs the first signal, the first promoter starts second feature and destroys element, and second feature signal is not
It exports again；After inputting third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input second
After signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature signal；
(4) when signal input sequence is 2-1-3,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, output second
Characteristic signal；After inputting the first signal, since the first circuit is destroyed, fisrt feature signal is no longer exported；Input third signal
Afterwards, unchanged；
After second cell inputs second signal, the second promoter starts fisrt feature and destroys element, and fisrt feature signal is not
It exports again；After inputting the first signal, the first promoter starts second feature and destroys element, and second feature signal no longer exports；It is defeated
After entering third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
(5) when signal input sequence is 2-3-1,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, output second
Characteristic signal；It is unchanged after inputting third signal；After inputting the first signal, since the first circuit is destroyed, first is no longer exported
Characteristic signal；
After second cell inputs second signal, the second promoter starts fisrt feature and destroys element, and fisrt feature signal is not
It exports again；After inputting third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input first
After signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature signal；
(6) when signal input sequence is 3-1-2,
It is unchanged after first cell inputs third signal；After inputting the first signal, the first promoter starts the first circuit,
Second circuit is destroyed, and exports fisrt feature signal；After inputting second signal, since second circuit is destroyed, no longer output the
Two characteristic signals；
After second cell inputs third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
After inputting the first signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature letter
Number；After inputting second signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, and it is special still to export first
Reference number；
(7) when signal input sequence is 3-2-1,
It is unchanged after first cell inputs third signal；After inputting second signal, the second promoter starts second circuit,
First circuit is destroyed, and exports second feature signal；After inputting the first signal, since the first circuit is destroyed, no longer output the
One characteristic signal；
After second cell inputs third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
After inputting second signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature letter
Number；After inputting the first signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, and it is special still to export second
Reference number.
Correspondingly, the results are shown in Table 2 for the output of sequence identifier.
The 4th replaced output result of circuit of 2 second cell of table
Above-mentioned bacterial cell sequence identifier, the 5th circuit of the second cell can be replaced only by third signal sensitivity
Four circuit damage elements composition.It is not necessarily to promoter and terminator, only needs the sensitive 4th circuit damage element of a third signal,
As shown in Figure 5.
The replaced bacterial cell sequence identifier of 5th circuit, the method for identification signal sequence are as follows:
(1) when original state,
First the first circuit of cell, second circuit do not work, non-output signal；
Only tertiary circuit works second cell, exports fisrt feature signal and second feature signal；
(2) when signal input sequence is 1-2-3,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, output first
Characteristic signal；After inputting second signal, since second circuit is destroyed, second feature signal is no longer exported；Input third signal
Afterwards, unchanged；
After second cell inputs the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal is not
It exports again；After inputting second signal, the second promoter starts second feature and destroys element, and second feature signal no longer exports, defeated
After entering third signal, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is destroyed；
(3) when signal input sequence is 1-3-2,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, output first
Characteristic signal；It is unchanged after inputting third signal；After inputting second signal, since second circuit is destroyed, second is no longer exported
Characteristic signal；
After second cell inputs the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal is not
It exports again；After inputting third signal, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is destroyed；Input the
After binary signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature signal；
(4) when signal input sequence is 2-1-3,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, output second
Characteristic signal；After inputting the first signal, since the first circuit is destroyed, fisrt feature signal is no longer exported；Input third signal
Afterwards, unchanged；
After second cell inputs second signal, the second promoter starts second feature and destroys element, and second feature signal is not
It exports again；After inputting the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal no longer exports；It is defeated
After entering third signal, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is destroyed；
(5) when signal input sequence is 2-3-1,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, output second
Characteristic signal；It is unchanged after inputting third signal；After inputting the first signal, since the first circuit is destroyed, first is no longer exported
Characteristic signal；
After second cell inputs second signal, the second promoter starts second feature and destroys element, and second feature signal is not
It exports again；After inputting third signal, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is destroyed；Input the
After one signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature signal；
(6) when signal input sequence is 3-1-2,
It is unchanged after first cell inputs third signal；After inputting the first signal, the first promoter starts the first circuit,
Second circuit is destroyed, and exports fisrt feature signal；After inputting second signal, since second circuit is destroyed, no longer output the
Two characteristic signals；
After second cell inputs third signal, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is broken
It is bad；After inputting the first signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, and it is special still to export first
Reference number；After inputting second signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output the
Two characteristic signals；
(7) when signal input sequence is 3-2-1,
It is unchanged after first cell inputs third signal；After inputting second signal, the second promoter starts second circuit,
First circuit is destroyed, and exports second feature signal；After inputting the first signal, since the first circuit is destroyed, no longer output the
One characteristic signal；
After second cell inputs third signal, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is broken
It is bad；After inputting second signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, and it is special still to export second
Reference number；After inputting the first signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output the
One characteristic signal.
Correspondingly, the results are shown in Table 3 for the output of sequence identifier.
The 5th replaced output result of circuit of 3 second cell of table
The present invention also provides the cytocomputers comprising several above-mentioned bacterial cell sequence identifiers.
Compared with prior art, the invention has the benefit that
Bacterial cell sequence identifier provided by the invention is allowed to no longer work from now on, come by destroying circuit or element
Tracer signal input sequence, and the finger to three kinds of six kinds of signal different input sequences is realized by the output combination of two kinds of cells
Show, can be used for biocomputer, biosensor etc. and need to determine in the related application of input sequence.
Detailed description of the invention
In order to illustrate the technical solutions in the embodiments of the present application or in the prior art more clearly, below will be to institute in embodiment
Attached drawing to be used is needed to be briefly described, it should be apparent that, the accompanying drawings in the following description is only one recorded in the present invention
A little embodiments are also possible to obtain other drawings based on these drawings for those of ordinary skill in the art.
Fig. 1 is the genetic circuit figure of prior art cell counter；
Fig. 2 is the genetic circuit figure of the first cell of the invention；
Fig. 3 is the genetic circuit figure of the second cell of the invention；
Fig. 4 is the replaced genetic circuit figure of the 4th circuit of the second cell of the invention；
Fig. 5 is the replaced genetic circuit figure of the 5th circuit of the second cell of the invention；
Fig. 6 is the genetic circuit figure of the first cell of the embodiment of the present invention 1；
Fig. 7 is the genetic circuit figure of the second cell of the embodiment of the present invention 1；
Fig. 8 is the genetic circuit figure of the first cell of the embodiment of the present invention 2；
Fig. 9 is the genetic circuit figure of the second cell of the embodiment of the present invention 2；
Figure 10 is the genetic circuit figure of the first cell of the embodiment of the present invention 3；
Figure 11 is the genetic circuit figure of the second cell of the embodiment of the present invention 3.
Specific embodiment
In order to make those skilled in the art more fully understand technical solution of the present invention, below in conjunction with attached drawing and implementation
Example is further detailed the present invention.
Embodiment 1
This example is used for aTc, and the input sequence of tri- kinds of signals of Ara, IPTG is indicated, using green fluorescence and red
Fluorescence is constructed using CRISPR/Cas9 system as characteristic signal and destroys element.CRISPR/Cas9(clustered
Regularly interspaced short palindromic repeats/CRISPR-associated9system) system
It is established on the basis of II type CRISPR/Cas system within 2012.The system is specific in a kind of editor target cell of RNA mediation
Gene order system, applied in the organisms such as human cell, yeast, bacterium and arabidopsis.The system by
Two parts composition: a part is can to cut gene " scalpel " Cas9 albumen, another part be in tow " scalpel " thin
Pinpoint guide RNA (sgRNA) in born of the same parents.After sgRNA positioning, Cas9 albumen forms DNA double chain fracture in the genome
(DSB), subsequent cell realizes that efficient gene knocks out, to reach by means of itself repair system, such as non-homologous end joining
To the effect for destroying Genetic elements.
The selection of promoter and characteristic signal is as follows:
First promoter: the promoter P of the first signal aTc startingtet
Second promoter: the promoter P of second signal Ara startingBAD
Third promoter: the promoter P of third signal IPTG startinglac
Fisrt feature signal: green fluorescent protein GFP
Second feature signal: red fluorescent protein RFP
For convenient for operating, all circuits of the first cell and the second cell are implemented on plasmid, sgRNA passes through positioning matter
Grain replication orgin realizes destruction of the Cas9 albumen to entire circuit, realizes that Cas9 albumen breaks discrete component by gene location
Bad, the specific genetic circuit building of the first cell is as shown in fig. 6, the specific genetic circuit building of the second cell is as shown in Figure 7.
The course of work of the first cell and the second cell is as shown in table 4 (because the first cell is to the under various input sequences
Three signal IPTG are reactionless, therefore the course of work after IPTG is added is omitted in the first cell).
First cell, the second cell work process under the various input sequences of table 4
Correspondingly, the output of sequence identifier is as shown in table 5.
The output of 5 example 1 of table
In this sequence identifier, sgRNA design follows CRISPR/Cas9 system requirements, should be according to each cell specific works
Situation is selected or building can ensure that E.coli bacterial strain or other bacterial strains that promoter related and CRISPR/Cas9 works.First is thin
Born of the same parents, the second cell should select identical or coherent signal inducing expression time similar strain.Constitutive promoter is selected in corresponding bacterium
The promoter worked normally in strain.Each intracellular different plasmid circuits should select different resistances to indicate as screening.Plasmid construction
And it is transformed into host cell and follows common molecular clone operations method.Signal input is using conventional derivational expression method.First is thin
Born of the same parents, the second cell are separately cultivated, while input signal are added.As a result detecting optional fluorescence microscope, flow cytometer etc. can
The equipment for detecting fluorescin.
Embodiment 2
This example is used for aTc, and the input sequence of tri- kinds of signals of Ara, IPTG is indicated, using green fluorescence and red
Fluorescence pinpoints recombination enzyme system building using DNA and destroys element as characteristic signal.DNA fixed point recombinase can identify specifically
The site DNA simultaneously mediates the DNA between recognition site to recombinate, and the direction of recognition site will determine the form of recombination, such as deletion or inversion.
DNA fragmentation be deleted or inversion after, original function can not be played, achieve the purpose that realize that circuit or element are destroyed.It is common
DNA fixed point recombination enzyme system include Cre/loxP, FLP/FRT, Bxb1/attB/attP system etc..
The selection of promoter and characteristic signal is as follows:
First promoter: the promoter P of the first signal aTc startingtet
Second promoter: the promoter P of second signal Ara startingBAD
Third promoter: the promoter P of third signal IPTG startinglac
Fisrt feature signal: green fluorescent protein GFP
Second feature signal: red fluorescent protein RFP
For convenient for operating, all circuits of the first cell and the second cell are implemented on plasmid, it is logical that DNA pinpoints recombinase
The destruction for deleting circuit realization to entire circuit is crossed, the destruction by deletion gene realization to discrete component, the first of this example
The specific genetic circuit of cell constructs as shown in figure 8, the specific genetic circuit building of the second cell is as shown in Figure 9.
The course of work of the first cell and the second cell is as shown in table 6 (because the first cell is to the under various input sequences
Three signal IPTG are reactionless, therefore the course of work after IPTG is added is omitted in the first cell).
First cell, the second cell work process under the various input sequences of table 6
Correspondingly, the output of sequence identifier is as shown in table 7.
The output of 7 example 2 of table
In this sequence identifier, should select or construct according to each cell specific works situation can ensure that it is promoter related and again
The E.coli bacterial strain or other bacterial strains of group enzyme system work.First cell, the second cell should select the induction of identical or coherent signal
Expression time similar strain.Constitutive promoter selects the promoter worked normally in corresponding bacterial strain.Plasmid construction and conversion
Enter host cell and follows common molecular clone operations method.Each intracellular different plasmid circuits should select different resistances as screening
Mark.Signal input is using conventional derivational expression method.First cell, the second cell are separately cultivated, while adding input signal.
As a result the equipment that the detectable fluorescins such as fluorescence microscope, flow cytometer can be selected is detected.
Embodiment 3
The 5th circuit of the second cell of this example is used for using flexible structure to aTc, Ara, three kinds of signals of temperature (37 DEG C)
Input sequence is indicated, using green fluorescence and red fluorescence as characteristic signal, using CRISPR/Cas9 system and temperature
Sensitive replication origin building destroys element.Temperature sensitive replication origin repA101ts can lose function at high temperature (37 DEG C or more)
Can, lead to entire plasmid not reproducible, to destroy the function of circuit on plasmid.
The selection of promoter and characteristic signal is as follows:
First promoter: the promoter P of the first signal aTc startingtet
Second promoter: the promoter P of second signal Ara startingBAD
The sensitive 4th circuit damage element of third signal: the temperature sensitive temperature sensitive replication origin of third signal
repA101ts
Fisrt feature signal: green fluorescent protein GFP
Second feature signal: red fluorescent protein RFP
For convenient for operating, all circuits of the first cell and the second cell are implemented on plasmid, sgRNA passes through positioning matter
Grain replication orgin realizes destruction of the Cas9 albumen to entire circuit, realizes that Cas9 albumen breaks discrete component by gene location
Bad, as shown in Figure 10, the building of the second cell physical circuit is as shown in figure 11 for the first cell physical circuit building of this example.
The course of work of the first cell and the second cell is as shown in table 8 (because the first cell is to the under various input sequences
Three 37 DEG C of signal temperature are reactionless, therefore the course of work that temperature is increased to after 37 DEG C is omitted in the first cell).
First cell, the second cell work process under the various input sequences of table 8
Correspondingly, the output of sequence identifier is as shown in table 8.
The output of 8 example 3 of table
In this sequence identifier, sgRNA design follows CRISPR/Cas9 system requirements, should be according to each cell specific works
Situation is selected or building can ensure that E.coli bacterial strain or other bacterial strains that promoter related and CRISPR/Cas9 works.First is thin
Born of the same parents, the second cell should select identical or coherent signal inducing expression time similar strain.Constitutive promoter is selected in corresponding bacterium
The promoter worked normally in strain.Each intracellular different plasmid circuits should select different resistances to indicate as screening.Plasmid construction
And it is transformed into host cell and follows common molecular clone operations method.Signal input is considered using conventional derivational expression method
To the presence of temperature sensitive promoter, the case where input is 37 DEG C is removed, other situations all should (25 DEG C -30 DEG C) progress at low temperature
Bacteria Culture need to extend 2 to 4 hours by incubation time on a regular basis.First cell, the second cell are separately cultivated, simultaneously
Add input signal.As a result the equipment that the detectable fluorescins such as fluorescence microscope, flow cytometer can be selected is detected.
It is above that certain exemplary embodiments of the invention are only described by way of explanation, undoubtedly, for ability
The those of ordinary skill in domain without departing from the spirit and scope of the present invention can be with a variety of different modes to institute
The embodiment of description is modified.Therefore, foregoing description is regarded as illustrative in nature, and should not be construed as wanting right of the present invention
Ask the limitation of protection scope.
Claims (7)
1. a kind of bacterial cell sequence identifier, which is characterized in that its by the two kinds of cell compositions of the first cell and the second cell,
In, the sequence of the first cell the first signal and the second signal for identification, the second cell third signal and first, for identification
The sequence of binary signal；
The genetic circuit of first cell includes the first circuit and second circuit；Wherein,
First circuit is successively destroyed element, fisrt feature signal and terminator and is formed by the first promoter, second circuit；
Second circuit is successively made of the second promoter, the first circuit damage element, second feature signal and terminator；
The genetic circuit of second cell includes tertiary circuit, the 4th circuit and the 5th circuit；Wherein,
Tertiary circuit is successively made of constitutive promoter, fisrt feature signal, second feature signal and terminator；
4th circuit successively destroys element, terminator, the second promoter, second feature by the first promoter, fisrt feature and destroys
Element and terminator composition；
5th circuit is successively made of third promoter, the 4th circuit damage element and terminator；
Wherein, the first promoter is started by the first signal enabling, the second promoter by second signal, and third promoter is believed by third
Number starting, circuit damage element can destroy the work of entire circuit, and feature destroys the work of element only destructive characteristics signal.
2. bacterial cell sequence identifier according to claim 1, which is characterized in that the method for its identification signal sequence
Are as follows:
(1) when original state,
First the first circuit of cell, second circuit do not work, non-output signal；
Only tertiary circuit works second cell, exports fisrt feature signal and second feature signal；
(2) when signal input sequence is 1-2-3,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, and exports fisrt feature
Signal；After inputting second signal, since second circuit is destroyed, second feature signal is no longer exported；After inputting third signal, nothing
Variation；
After second cell inputs the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal is no longer defeated
Out；After inputting second signal, the second promoter starts second feature and destroys element, and second feature signal no longer exports, input the
After three signals, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
(3) when signal input sequence is 1-3-2,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, and exports fisrt feature
Signal；It is unchanged after inputting third signal；After inputting second signal, since second circuit is destroyed, second feature is no longer exported
Signal；
After second cell inputs the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal is no longer defeated
Out；After inputting third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input second signal
Afterwards, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature signal；
(4) when signal input sequence is 2-1-3,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, and exports second feature
Signal；After inputting the first signal, since the first circuit is destroyed, fisrt feature signal is no longer exported；After inputting third signal, nothing
Variation；
After second cell inputs second signal, the second promoter starts second feature and destroys element, and second feature signal is no longer defeated
Out；After inputting the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal no longer exports；Input the
After three signals, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
(5) when signal input sequence is 2-3-1,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, and exports second feature
Signal；It is unchanged after inputting third signal；After inputting the first signal, since the first circuit is destroyed, fisrt feature is no longer exported
Signal；
After second cell inputs second signal, the second promoter starts second feature and destroys element, and second feature signal is no longer defeated
Out；After inputting third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input the first signal
Afterwards, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature signal；
(6) when signal input sequence is 3-1-2,
It is unchanged after first cell inputs third signal；After inputting the first signal, the first promoter the first circuit of starting, second
Circuit is destroyed, and exports fisrt feature signal；After inputting second signal, since second circuit is destroyed, it is special no longer to export second
Reference number；
After second cell inputs third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input
After first signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature signal；
After inputting second signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature letter
Number；
(7) when signal input sequence is 3-2-1,
It is unchanged after first cell inputs third signal；After inputting second signal, the second promoter starting second circuit, first
Circuit is destroyed, and exports second feature signal；After inputting the first signal, since the first circuit is destroyed, it is special no longer to export first
Reference number；
After second cell inputs third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input
After second signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature signal；
After inputting the first signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature letter
Number.
3. bacterial cell sequence identifier according to claim 1, which is characterized in that the 4th circuit of the second cell is replaced
Element and termination are destroyed successively to destroy element, terminator, the second promoter, fisrt feature by the first promoter, second feature
Son composition.
4. bacterial cell sequence identifier according to claim 3, which is characterized in that the method for its identification signal sequence
Are as follows:
(1) when original state,
First the first circuit of cell, second circuit do not work, non-output signal；
Only tertiary circuit works second cell, exports fisrt feature signal and second feature signal；
(2) when signal input sequence is 1-2-3,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, and exports fisrt feature
Signal；After inputting second signal, since second circuit is destroyed, second feature signal is no longer exported；After inputting third signal, nothing
Variation；
After second cell inputs the first signal, the first promoter starts second feature and destroys element, and second feature signal is no longer defeated
Out；After inputting second signal, the second promoter starts fisrt feature and destroys element, and fisrt feature signal no longer exports, input the
After three signals, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
(3) when signal input sequence is 1-3-2,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, and exports fisrt feature
Signal；It is unchanged after inputting third signal；After inputting second signal, since second circuit is destroyed, second feature is no longer exported
Signal；
After second cell inputs the first signal, the first promoter starts second feature and destroys element, and second feature signal is no longer defeated
Out；After inputting third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input second signal
Afterwards, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature signal；
(4) when signal input sequence is 2-1-3,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, and exports second feature
Signal；After inputting the first signal, since the first circuit is destroyed, fisrt feature signal is no longer exported；After inputting third signal, nothing
Variation；
After second cell inputs second signal, the second promoter starts fisrt feature and destroys element, and fisrt feature signal is no longer defeated
Out；After inputting the first signal, the first promoter starts second feature and destroys element, and second feature signal no longer exports；Input the
After three signals, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；
(5) when signal input sequence is 2-3-1,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, and exports second feature
Signal；It is unchanged after inputting third signal；After inputting the first signal, since the first circuit is destroyed, fisrt feature is no longer exported
Signal；
After second cell inputs second signal, the second promoter starts fisrt feature and destroys element, and fisrt feature signal is no longer defeated
Out；After inputting third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input the first signal
Afterwards, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature signal；
(6) when signal input sequence is 3-1-2,
It is unchanged after first cell inputs third signal；After inputting the first signal, the first promoter the first circuit of starting, second
Circuit is destroyed, and exports fisrt feature signal；After inputting second signal, since second circuit is destroyed, it is special no longer to export second
Reference number；
After second cell inputs third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input
After first signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature signal；
After inputting second signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature letter
Number；
(7) when signal input sequence is 3-2-1,
It is unchanged after first cell inputs third signal；After inputting second signal, the second promoter starting second circuit, first
Circuit is destroyed, and exports second feature signal；After inputting the first signal, since the first circuit is destroyed, it is special no longer to export first
Reference number；
After second cell inputs third signal, third promoter starts the 4th circuit damage element, and the 4th circuit is destroyed；Input
After second signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature signal；
After inputting the first signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature letter
Number.
5. bacterial cell sequence identifier according to claim 1, which is characterized in that the 5th circuit of the second cell is replaced
To be only made of the sensitive 4th circuit damage element of third signal.
6. bacterial cell sequence identifier according to claim 5, which is characterized in that the method for its identification signal sequence
Are as follows:
(1) when original state,
First the first circuit of cell, second circuit do not work, non-output signal；
Only tertiary circuit works second cell, exports fisrt feature signal and second feature signal；
(2) when signal input sequence is 1-2-3,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, and exports fisrt feature
Signal；After inputting second signal, since second circuit is destroyed, second feature signal is no longer exported；After inputting third signal, nothing
Variation；
After second cell inputs the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal is no longer defeated
Out；After inputting second signal, the second promoter starts second feature and destroys element, and second feature signal no longer exports, input the
After three signals, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is destroyed；
(3) when signal input sequence is 1-3-2,
After first cell inputs the first signal, the first promoter starts the first circuit, and second circuit is destroyed, and exports fisrt feature
Signal；It is unchanged after inputting third signal；After inputting second signal, since second circuit is destroyed, second feature is no longer exported
Signal；
After second cell inputs the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal is no longer defeated
Out；After inputting third signal, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is destroyed；The second letter of input
After number, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature signal；
(4) when signal input sequence is 2-1-3,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, and exports second feature
Signal；After inputting the first signal, since the first circuit is destroyed, fisrt feature signal is no longer exported；After inputting third signal, nothing
Variation；
After second cell inputs second signal, the second promoter starts second feature and destroys element, and second feature signal is no longer defeated
Out；After inputting the first signal, the first promoter starts fisrt feature and destroys element, and fisrt feature signal no longer exports；Input the
After three signals, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is destroyed；
(5) when signal input sequence is 2-3-1,
After first cell inputs second signal, the second promoter starts second circuit, and the first circuit is destroyed, and exports second feature
Signal；It is unchanged after inputting third signal；After inputting the first signal, since the first circuit is destroyed, fisrt feature is no longer exported
Signal；
After second cell inputs second signal, the second promoter starts second feature and destroys element, and second feature signal is no longer defeated
Out；After inputting third signal, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is destroyed；The first letter of input
After number, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature signal；
(6) when signal input sequence is 3-1-2,
It is unchanged after first cell inputs third signal；After inputting the first signal, the first promoter the first circuit of starting, second
Circuit is destroyed, and exports fisrt feature signal；After inputting second signal, since second circuit is destroyed, it is special no longer to export second
Reference number；
After second cell inputs third signal, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is destroyed；It is defeated
After entering the first signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, still output fisrt feature letter
Number；After inputting second signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, and it is special still to export second
Reference number；
(7) when signal input sequence is 3-2-1,
It is unchanged after first cell inputs third signal；After inputting second signal, the second promoter starting second circuit, first
Circuit is destroyed, and exports second feature signal；After inputting the first signal, since the first circuit is destroyed, it is special no longer to export first
Reference number；
After second cell inputs third signal, the sensitive 4th circuit damage element manipulation of third signal, the 4th circuit is destroyed；It is defeated
After entering second signal, since the 4th circuit is destroyed, second feature is destroyed element and is not activated, still output second feature letter
Number；After inputting the first signal, since the 4th circuit is destroyed, fisrt feature is destroyed element and is not activated, and it is special still to export first
Reference number.
7. a kind of cytocomputer, which is characterized in that comprising several as bacterial cell as claimed in any one of claims 1 to 6 is suitable
Sequence identifier.
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