CN104131018A - Intracellular protein self-induction expression method - Google Patents
Intracellular protein self-induction expression method Download PDFInfo
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- CN104131018A CN104131018A CN201410373158.3A CN201410373158A CN104131018A CN 104131018 A CN104131018 A CN 104131018A CN 201410373158 A CN201410373158 A CN 201410373158A CN 104131018 A CN104131018 A CN 104131018A
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Abstract
The invention relates to an intracellular protein self-induction expression method, and relates to the field of genetic engineering. A promoter and a structural gene corresponding to vibrio fischeri quorum sensing are cloned into a plasmid, so that a self-induction gene circuit is constructed, and the gene circuit is lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR. Automatic large-scale induction expression of subsequent target protein in a relatively high thalli concentration can be realized without depending an inducer through self positive feedback of the circuit, and separation of a cell growing period and product expression time is realized, so that product yield is improved. Because no inducer is added, cost is saved and operation process is simplified. Additionally, through special promoter design, the circuit has application prospect for detecting heavy metal ions, agricultural pollution bacteria, environmental change and the like.
Description
Technical field
The present invention relates to genetically engineered field, specifically, relate to a kind of method that can realize the expression of intracellular protein self-induction.
Background technology
Since the nineties in 20th century, a large amount of research work show, in bacterium, ubiquity the information interchange between bacterium and bacterium, and is mediating the adjusting of a series of physiological behaviors, and this information interchange is the quorum sensing of bacterium.General regulation process is: bacterium utilizes in signaling molecule perception surrounding environment the variation of the cell colony density of self or other bacteriums, and signaling molecule increases along with the increase of population density, when population density reaches certain threshold value, signaling molecule will start the expression of specific gene in thalline, behavior between change and coordination cell, thereby realize some physiological function and regulation mechanism that single bacterium cannot complete, present certain physiological property.
Research to ocean Fei Shi vibrios (Vibrio fischeri) quorum sensing genes involved starts from nineteen eighty-three, to study up to now the most thorough quorum sensing system, therefore its luxI/luxR two-component system is regarded as the modular system of gram negative bacteria quorum sensing, luxI genes encoding signaling molecule AHL synthetic enzyme wherein, luxR genes encoding regulates albumen.When low cell density, each cell is the composing type AHL of synthetic basal level only, produces immune response to avoid being identified by host cell; Along with cell quantity increases gradually, the AHL that can free in and out cell accumulates inside and outside cell, when sufficiently high cell density makes AHL reach threshold level (concentration is 10nmol/L), signaling molecule can with bacterium in LuxR regulate protein binding to form dimer mixture, this mixture is combined in the promotor lux box region of goal gene, can activate the expression of downstream luciferase gene (luxCDABE) on the one hand, cause vibrios luminous; On the other hand, start rapidly the luxI genetic expression of coding AHL synthetic enzyme, induction AHL self produces, and promotes a large amount of release of AHL signaling molecule and forms LuxR ∷ AHL mixture, forms positive regeeration, and above-mentioned QS system is constantly amplified.
Conventionally, in the optimization that fermentation process is carried out, mainly put forth effort on the highest possibility productive rate that obtains product, to a kind of solution of this problem, be to obtain high as far as possible cell concentration, usually in the process of fermentation, when cell concentration reaches certain value, the material that starts to add some inductors or can be metabolized to inductor, make product start to synthesize under higher cell concentration, as Chinese patent 201010221511.8 discloses a kind of fermentation process that utilizes lactose-induced pMFH carrier Restruction albumen, the method is added during the fermentation after lactose-induced and is added glycerine as carbon source again, in the time of to raising biomass, also improve the expression amount of recombinant protein.Above-mentioned method need to be monitored the upgrowth situation of bacterium, to add inductor in suitable vegetative period, sometimes even need to change the culture condition in later stage, therefore expectation is, can be by a kind of design of gene pathway, in the growth early stage of recipient bacterium, more energy nutrient is used to the growth of thalline, after reaching certain cell concentration, product will be synthetic by automatically a large amount of inductions, the energy nutrient that now flows into thalli growth will tail off, and thalline is used for the synthetic of product more energy nutrient, makes products collection efficiency reach a higher value.
Summary of the invention
The object of this invention is to provide a kind of method that intracellular protein self-induction is expressed.
Concrete steps of the present invention are as follows:
The Fei Shi vibrios corresponding promotor of (Vibrio fischeri) quorum sensing and structure gene are cloned in plasmid, built a self-induced gene pathway, this path is: lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR, it does not rely on inductor just can realize by the positive regeeration of path itself follow-up target protein a large amount of abduction deliverings automatically under higher cell concentration, realize separating of phase of cell growth and Product Expression time, and then improved products collection efficiency.
Described gene pathway, contains luxR, the luxI and promotor lux pR and the lux pL that are cloned in Fei Shi vibrios (Vibrio fischeri);
Contained luxR is not limited to the luxR of wild-type Fei Shi vibrios, and it can be through the luxR of improvement or have other gene orders with the luxR identity function of wild-type Fei Shi vibrios.
Contained luxI is not limited to the luxI of wild-type Fei Shi vibrios, and it can be through the luxI of improvement or have other gene orders with the luxI identity function of wild-type Fei Shi vibrios.
Contained lux pR is not limited to the lux pR of wild-type Fei Shi vibrios, and it can be lux pR or other special promoters through improvement.
Contained lux pL is not limited to the lux pL of wild-type Fei Shi vibrios, and it can be lux pL or other special promoters through improvement.
Described gene pathway, just can realize the abduction delivering of follow-up gene without the inductor adding as IPTG etc.
The present invention has built the gene pathway of a simulation quorum sensing, it is imported in recipient bacterium, make this receptor cell have constructed quorum sensing system, be about to Fei Shi vibrios quorum sensing genes involved in conjunction with specific promotor, carrier construction also imports intestinal bacteria, can realize artificial constructed quorum sensing path.The existence of quorum sensing gene positive feedback mechanism, make follow-up target protein gene a large amount of abduction deliverings automatically under a higher cell concentration, realized separating of phase of cell growth and Product Expression time, and then improved products collection efficiency.And by special promotor, design, this path also can be applicable to the aspects such as testing environment heavy metal contamination, testing environment variation and agricultural pollution bacterium.
The invention has the advantages that on the one hand owing to need not adding inductor, saved cost and simplified the operation course, can also design by special promotor on the other hand, make path possess application prospect at aspects such as detecting heavy metal ion, agricultural pollution bacterium, testing environment variation.
Accompanying drawing explanation
Fig. 1 is the plasmid spectrogram of self-induction path.
Fig. 2 is the fluorescence curve of path.
Embodiment
Embodiment mono-:
The structure of biological brick element lux pR-RBS-luxI-TT
Extract plasmid pSB1A2-RBS-luxI-TT; Double digestion through XbaI/PstI produces the rear end Insert Fragment RBS-luxI-TT of about 800bp and the plasmid skeleton pSB1A2 of about 2000bp, and glue reclaims wherein rear end Insert Fragment band RBS-luxI-TT; Extract plasmid pSB1A2-lux pR simultaneously, through SpeI/PstI double digestion, generate the rear end carrier segments pSB1A2-lux pR of about 2000bp; Connect above-mentioned two fragments, be transformed in DH5 ɑ, be coated on the LB culture medium flat plate that contains penbritin, 37 ℃ of cultivations, second day is chosen single bacterium colony, and 14h is cultivated in 37 ℃ of LB liquid nutrient medium 200r/min concussions, extract plasmid and carry out EcoRI and PstI double digestion and EcoRI single endonuclease digestion, the checking of race glue, the stripe size of single endonuclease digestion is about 3000bp, and the band of double digestion is respectively the object fragment lux pR-RBS-luxI-TT of about 900bp and the plasmid skeleton pSB1A2 of 2000bp left and right.This project bacterium is in-20 ℃ of preservations, with plasmid pSB1A2-lux pR-RBS-luxI-TT.
Embodiment bis-:
The structure of biological brick element lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR
Extract plasmid pSB1A2-lux pL-RBS-luxR-TT-lux pR, through EcoRI/XbaI double digestion, produce the front end carrier segments lux pL-RBS-luxR-TT-lux pR-pSB1A2 of 3000bp.Extract plasmid pSB1A2-lux pR-RBS-luxI-TT simultaneously, through EcoRI/SpeI double digestion, produce the front end Insert Fragment lux pR-RBS-luxI-TT of about 900bp and the plasmid skeleton pSB1A2 of 2000bp, run glue and reclaim front end Insert Fragment lux pR-RBS-luxI-TT; Connect above-mentioned front end carrier segments and front end Insert Fragment, be transformed in DH5 ɑ, be coated on the LB culture medium flat plate that contains penbritin, 37 ℃ of cultivations, second day is chosen single bacterium colony, 14h is cultivated in 37 ℃ of LB liquid nutrient medium 200r/min concussions, extract plasmid and carry out EcoRI and PstI double digestion and EcoRI single endonuclease digestion, the checking of race glue, the stripe size of single endonuclease digestion is about 4000bp, and the band of double digestion is respectively the plasmid skeleton pSB1A2 of about 2000bp and the object fragment lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR of about 2000bp.This project bacterium is in-20 ℃ of preservations, with plasmid pSB1A2-lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR.
Embodiment tri-:
The structure of biological brick element RBS-EGFP-TT
Extract plasmid pSB1A2-RBS, through SpeI/PstI double digestion, produce the rear end carrier segments pSB1A2-RBS of about 2000bp; Extract plasmid pSB1A2-EGFP-TT simultaneously, through XbaI/PstI double digestion, produce the plasmid skeleton of about 2000bp and the rear end Insert Fragment EGFP-TT of about 800bp, run glue and reclaim rear end Insert Fragment EGFP-TT; Connect above-mentioned rear end carrier and rear end Insert Fragment, be transformed in DH5 ɑ, be coated on the LB culture medium flat plate that contains penbritin, 37 ℃ of cultivations, second day is chosen single bacterium colony, and 37 ℃ of LB liquid nutrient medium 200r/min cultivate 14h, extract plasmid and carry out EcoRI and PstI double digestion and EcoRI single endonuclease digestion, the checking of race glue, the stripe size of single endonuclease digestion is about 2900bp, and the band of double digestion is respectively the plasmid skeleton pSB1A2 of about 2000bp and the object fragment RBS-EGFP-TT of about 900bp.This project bacterium is in-20 ℃ of preservations, with plasmid pSB1A2-RBS-EGFP-TT.
Embodiment tetra-:
The structure of self-induction path biological brick element lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR-RBS-EGFP-TT
Extract plasmid pSB1A2-lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR, through SpeI/PstI double digestion, obtain the rear end carrier segments pSB1A2-lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR of about 4000bp; Extract plasmid pSB1A2-RBS-EGFP-TT simultaneously, through XbaI/PstI double digestion, obtain the rear end Insert Fragment RBS-EGFP-TT of about 900bp and the plasmid skeleton pSB1A2 of about 2000bp, glue reclaims rear end Insert Fragment.Connect above-mentioned rear end Insert Fragment RBS-EGFP-TT and rear end carrier segments pSB1A2-lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR, be transformed in DH5 ɑ, be coated on the LB culture medium flat plate that contains penbritin, 37 ℃ of cultivations, second day is chosen single bacterium colony, 37 ℃ of LB liquid nutrient medium 200r/min cultivate 14h, extract plasmid and carry out EcoRI and PstI double digestion and EcoRI single endonuclease digestion, the checking of race glue, the stripe size of single endonuclease digestion is about 4900bp, the band of double digestion is respectively the plasmid skeleton pSB1A2 of 2000bp and the object fragment lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR-RBS-EGFP-TT of 2900bp.This project bacterium is in-20 ℃ of preservations, with plasmid pSB1A2-lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR-RBS-EGFP-TT.
Embodiment five:
The structure of control group biological brick element lux pL-RBS-luxR-TT-lux pR-RBS-EGFP-TT
Extract plasmid pSB1A2-lux pL-RBS-luxR-TT-lux pR, through EcoRI/SpeI double digestion, obtain the front end Insert Fragment lux pL-RBS-luxR-TT-lux pR of about 1200bp and the plasmid skeleton pSB1A2 of about 2000bp, run glue and reclaim front end Insert Fragment, extract plasmid pSB1A2-RBS-EGFP-TT simultaneously, through EcoRI/XbaI double digestion, obtain being about the front end carrier segments RBS-EGFP-TT-pSB1A2 of 2900bp, connect above-mentioned front end Insert Fragment lux pL-RBS-luxR-TT-lux pR and front end carrier segments RBS-EGFP-TT-pSB1A2, be transformed in DH5 ɑ, be coated on the LB culture medium flat plate that contains penbritin, 37 ℃ of cultivations, second day is chosen single bacterium colony, 37 ℃ of LB liquid nutrient medium 200r/min cultivate 14h, extract plasmid and carry out EcoRI and PstI double digestion and EcoRI single endonuclease digestion, the checking of race glue, the stripe size of single endonuclease digestion is about 4000bp, the band of double digestion is respectively the plasmid skeleton pSB1A2 of about 2000bp and the object fragment lux pL-RBS-luxR-TT-lux pR-RBS-EGFP-TT of 2000bp.This project bacterium is in-20 ℃ of preservations, with plasmid pSB1A2-lux pL-RBS-luxR-TT-lux pR-RBS-EGFP-TT.
Embodiment six:
The mensuration of fluorescence curve
(1) thalline activation: inoculate two parts of each 100 μ L of self-induction path bacterium liquid portion and control group bacterium liquid from glycerine pipe in 20mL LB substratum, add the penbritin of the 50mg/ml of 20 μ L simultaneously.37 ℃, the about 12h switching of 200r/min concussion activation.
(2) inoculation culture: respectively get 250 μ L bacterium liquid after activation and transfer in the 250mL Erlenmeyer flask of 50mL LB substratum is housed, the penbritin that simultaneously adds the 50mg/ml of 50 μ l, control group a copy of it is plus signal molecule AHL after switching 2h, as positive controls, signaling molecule final concentration is 200nmol/L, and another part of control group is as negative control group.37 ℃, 200r/min shakes cultivation.
(3) mensuration of fluorescence intensity: regularly get appropriate bacterium liquid every 1～2h, ice bath 10min, then 4 ℃, 5000r/min, the centrifugal lower collection thalline of 10min, and resuspended with the PBS damping fluid of 600 μ l ice precoolings, recentrifuge, with resuspended with the PBS damping fluid of original bacterium liquid measure same volume, ice bath or 4 ℃ of Refrigerator stores are to be measured.After dilution suitable multiple, utilize the fluorescence intensity of fluorescence spectrophotometer measurement green fluorescent protein.Excitation wavelength is 480nm, and emission wavelength is 515nm.Reclaim bacterium liquid and survey its A
600value.Utilize fluorescence intensity to analyze experimental result, fluorescent value=fluorescence intensity/A
600.
(4) draw luminosity curve: take the time as X-coordinate, take fluorescent value as ordinate zou drafting luminosity curve.
(5) result: compare with positive controls, when 7～8h, just there is the rising of obvious fluorescent value in the fluorescence curve of self-induction path (experimental group), and positive controls is in the existing trend rising of 3～4h, this is to have reached startup threshold value due to signaling molecule that control group adds, started at the first sampling follow-up EGFP genetic expression, and expression amount is along with the time is constantly accumulated, and experimental group concentration due to signaling molecule when former sub-sampling does not reach the threshold value that starts EGFP genetic expression, the amount fluctuation of GFP one among a small circle in, until 7～8h, the concentration of signaling molecule is enough high, GFP expression amount just starts to rise, the path of illustrative experiment group can the follow-up albumen of retardation expression, under higher cell concentration, the great expression of albumen just can be activated.Compare with negative control group, the fluorescent value of experimental group is stabilized between 50000～55000, in logarithmic phase, even reach more than 60000, and negative control group at the fluorescent value of whole growth period between 200～800, the fluorescent protein expression amount that is self-induction path exceeds and has 60～70 times more than than negative control group, can realize a large amount of abduction deliverings of albumen after the adjusting of visible self-induction path by self positive regeeration.
The plasmid spectrogram of self-induction path is referring to Fig. 1, and the fluorescence curve of path is referring to Fig. 2.
Claims (7)
1. the method that intracellular protein self-induction is expressed, is characterized in that its concrete steps are as follows:
The Fei Shi vibrios corresponding promotor of (Vibrio fischeri) quorum sensing and structure gene are cloned in plasmid, built a self-induced gene pathway, this path is: lux pR-RBS-luxI-TT-lux pL-RBS-luxR-TT-lux pR, it does not rely on inductor just can realize by the positive regeeration of path itself follow-up target protein a large amount of abduction deliverings automatically under higher cell concentration, realize separating of phase of cell growth and Product Expression time, and then improved products collection efficiency.
2. a kind of method that intracellular protein self-induction is expressed as claimed in claim 1, is characterized in that described gene pathway, contains luxR, the luxI and promotor lux pR and the lux pL that are cloned in Fei Shi vibrios (Vibrio fischeri).
3. a kind of method that intracellular protein self-induction is expressed as claimed in claim 2, it is characterized in that contained luxR is the luxR of wild-type Fei Shi vibrios, or through the luxR of improvement, or there are other gene orders with the luxR identity function of wild-type Fei Shi vibrios.
4. a kind of method that intracellular protein self-induction is expressed as claimed in claim 2, it is characterized in that contained luxI is the luxI of wild-type Fei Shi vibrios, or through the luxI of improvement, or there are other gene orders with the luxI identity function of wild-type Fei Shi vibrios.
5. a kind of method that intracellular protein self-induction is expressed as claimed in claim 2, is characterized in that contained lux pR is the lux pR of wild-type Fei Shi vibrios, or the lux pR through improveing, or other special promoters.
6. a kind of method that intracellular protein self-induction is expressed as claimed in claim 2, is characterized in that contained lux pL is the lux pL of wild-type Fei Shi vibrios, or the lux pL through improveing, or other special promoters.
7. a kind of method that intracellular protein self-induction is expressed as claimed in claim 1, is characterized in that described gene pathway, without adding, just can realize the abduction delivering of follow-up gene as the inductor of IPTG.
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