CN112553239B - Genome rearrangement control system and method based on unnatural amino acid - Google Patents
Genome rearrangement control system and method based on unnatural amino acid Download PDFInfo
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
A system and method for genome rearrangement regulation based on unnatural amino acids, the system comprising: a tool pair for unnatural amino acid encoding comprising an unnatural aminoacyl-tRNA synthetase and a tRNA that has an anticodon paired with a stop codon in frame with the Cre recombinase encoding gene, where the stop codon is translated into an unnatural amino acid by the presence of the unnatural amino acid and the unnatural aminoacyl-tRNA synthetase; a regulatory means for controlling Cre recombinase expression comprising a Cre recombinase encoding gene comprising a stop codon in its reading frame for specifying an insertion site for an unnatural amino acid, the stop codon producing a full length functional Cre recombinase upon read-through which is capable of acting on the recombination site LoxP to thereby initiate genomic rearrangement and produce a truncated inactivated Cre recombinase upon read-through failure of the stop codon. The invention can safely, efficiently and accurately regulate the occurrence of the SCRaMble process, and has important application value in the fields of metabolic engineering, synthetic genome and other related synthetic biology.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a genome rearrangement control system and method based on unnatural amino acid.
Background
Synthetic genome is one of the most important research contents of synthetic biology, and provides a new view and approach for understanding the nature of vital activities and creating organisms that are beneficial to human needs. The synthetic yeast genome Sc2.0 program is a benchmarking program in the field of synthetic genomes, aimed at synthesizing all chromosomes of Saccharomyces cerevisiae and applying them in a number of aspects in scientific research and applications. The synthetic chromosome rearrangement control technology (SCRaMble, synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution) is a core element in the design of a synthetic yeast genome, and can induce the synthetic strain to generate genome diversity and phenotype universality, and the mechanism of action is that the genome is subjected to various types of structural variation such as deletion, inversion, repetition and the like under the action of Cre recombinase through the guidance of recombination sites LoxP. Based on the above variations, more complex genotype diversity can be achieved by continuously inducing the integration of genomic variations using the SCRaMble technique. Recent research results show that part of yeast strains after SCRaMble generation can improve the heterologous expression yield of small molecules and proteins, so that the method has potential great practical application value.
Accurate control of the SCRaMble process is critical for the production and maintenance of genotype strains with particular advantages. Earlier studies have shown that fusion of the estrogen binding domain EBD with Cre recombinase allows ligand-dependent control of its activity. Thus, cre-EBD fusion proteins have been widely used to perform ligand-dependent recombination reactions in mammalian cells and yeast. Specifically, in conventional recombinant techniques, the pSCW11-Cre-EBD plasmid is used to activate the occurrence of SCRaMble. The pSCW11 promoter is a sub-cell specific promoter that precisely produces a pulse of expressed recombinase during the life cycle of each sub-generation cell. The function of Cre recombinase is posttranscriptionally regulated by the Estradiol Binding Domain (EBD), which in the absence of estradiol isolates the fusion protein (Cre-EBD) in the cytoplasm. With the addition of estradiol, cre recombinase is transported into the nucleus and acts on the recombination site LoxP. However, the Cre recombinase in the above method has a problem of functional leakage, that is, growth defects in the synthetic yeast containing the plasmid can be observed without estradiol induction, which results in a decrease in stability of the synthetic chromosome.
Aiming at the defects of the traditional recombination technology, two upgrading technologies are developed at present, and the traditional SCRaMble system is modified, so that the function leakage condition is relieved to a certain extent.
One is a galactose-operated Cre-EBD system. The method adopts pGAL1 promoter to regulate the expression of Cre-EBD fusion protein, thereby regulating the occurrence of SCRaMble. The pGAL1 promoter is a galactose-induced promoter, which promotes the expression of a gene downstream of the promoter in the presence of galactose or a galactose analog, and inhibits the expression of the gene downstream in the presence of glucose. Cre-EBD is a fusion protein of Cre recombinase and EBD, and the presence of estradiol is required to bring the fusion protein into the nucleus for its function. Thus, the galactose-operated Cre-EBD system is a method of dual regulation of transcription and cell localization levels, requiring the simultaneous addition of galactose and estradiol to allow SCRaMble to occur.
The other is the optically controlled Cre system (L-SCRaMbLE system), which is a method of adjusting genome rearrangement based on illumination of a specific wavelength. The induction of recombinase activity under red light irradiation can be up to 179-fold, the degree of recombination depending on the induction time and the concentration of chromatin Phycocyanin (PCB). L-SCRaMLE was designed based on an abscisic protein in which the N-and C-terminus of Cre were fused to two heterologous proteins, namely, the chromatin-binding photoreceptor plant pigment B (PhyB) from Arabidopsis thaliana and its interacting factor PIF3. These two vegetable proteins, in the presence of PCB (chromogen of PhyB photoreceptors), reconstruct the functional Cre recombinase in dependence of the interaction of light. L-SCRaMble has very low basal activity in the absence of light and has a pronounced recombination capacity after red light exposure. And its recombination frequency can be fine-tuned in a dose-dependent manner in terms of PCB, time and light.
Despite the broad initial application prospects of SCRaMble, there are many potential challenges. The conventional method pSCW11-Cre-EBD plasmid has the problem of functional leakage, resulting in unstable genotype of the synthesized yeast without estradiol induction and after Cre induction is completed, thereby generating growth defects. Selective loss of plasmids by serial passage can increase the stability of the synthetic chromosome, but this approach comes at the cost of producing more replicates and potentially reducing the diversity of the SCRaMbLE cell population. If a related operation of plasmid loss is performed between each cycle, the iterative cycle of SCRaMble will be disturbed and make the operation cumbersome. In addition, estradiol acts as a potent inducer of SCRaMbLE, but it is a hormone that is toxic to humans at high concentrations, thereby causing potential safety problems for experimenters.
The galactose-operated Cre-EBD system, while having significant advantages over the conventional methods described above, residual galactose in the culture medium will result in sustained expression of the Cre-EBD fusion protein, resulting in instability of the genotype. Furthermore, previous studies found that protein fusion would result in an affected activity of Cre recombinase. Similar to the traditional methods, estradiol used in the procedure has hormonal activity and is toxic to the human body at elevated concentrations, thus causing potential safety problems for the experimenter. Thus, galactose-operated Cre-EBD systems still have certain drawbacks and deficiencies.
Compared with the traditional chemical induction method, the L-SCRaMLE has obvious advantages, and the system has very low basic activity under no light irradiation, so that the Cre recombinase function leakage is effectively reduced. L-SCRaMbLE relies on repeated red light pulses applied over a longer period of time, which effectively compensates for the drawbacks of using estradiol. Although L-SCRaMble is a good strategy for yeast. However, this method is still not an optimal choice for a larger range of subjects, such as mammalian cells, where the growth time is longer. In addition, the rearrangement efficiency of the method is far lower than that of the traditional method, and the method is complex in operation and requires special instruments.
In summary, a safe, universal method capable of fine-regulating Cre recombinase expression is needed to be developed on the premise of ensuring recombination efficiency, and is applied to SCRaMble expansion to more scale applications.
Disclosure of Invention
The invention provides a genome rearrangement regulating system and method based on unnatural amino acid, which can safely, efficiently and accurately regulate the occurrence of an SCRaMble process and has important application value in the fields of metabolic engineering, synthetic genome and other related synthetic biology.
According to a first aspect of the present invention there is provided a non-natural amino acid based genome rearrangement control system comprising:
a tool pair for coding an unnatural amino acid, the tool pair comprising an unnatural aminoacyl-tRNA synthetase and an acting tRNA thereof, where the anticodon of the tRNA is paired with a stop codon set in frame of a Cre recombinase-encoding gene, where the stop codon is translated into the unnatural amino acid by the presence of the unnatural amino acid and the action of the tool pair; and
a regulatory means for controlling Cre recombinase expression, the regulatory means comprising a Cre recombinase encoding gene comprising in its reading frame at least one set stop codon for an insertion site for an unnatural amino acid, producing a full length functional Cre recombinase upon reading through the stop codon and a truncated inactive Cre recombinase upon non-reading through the stop codon, said functional Cre recombinase being capable of acting on the recombination site LoxP to thereby initiate genome rearrangement.
In a preferred embodiment, the unnatural amino acid is O-methyl-L-tyrosine and the unnatural aminoacyl-tRNA synthetase is a methoxytyrosyl-tRNA synthetase.
In a preferred embodiment, the stop codon is a stop codon TAG substituted by the codon encoding leucine 14 at the N-terminus of the Cre recombinase, and the tRNA is a tRNA with an anticodon CUA.
According to a second aspect of the present invention there is provided a recombinant expression vector for use in producing the genomic rearrangement control system of the first aspect comprising:
a tool-paired recombinant expression vector comprising a vector backbone and coding elements paired with the tool; and
a recombinant expression vector for a regulatory tool comprising a vector backbone and coding elements for the regulatory tool.
In a preferred embodiment, the above tool-set recombinant expression vector further comprises a marker gene for leucine expression; the recombinant expression vector of the regulation tool also comprises a marker gene for expressing histidine.
In a preferred embodiment, the recombinant expression vector further comprises a reporter recombinant expression vector comprising a vector backbone and a reporter gene and a terminator positioned in front of the reporter gene and carrying LoxP recombination sites at the head and tail, wherein when rearrangement occurs, the terminator is deleted and the reporter gene is expressed normally.
In a preferred embodiment, the reporter gene is a GFP gene.
In a preferred embodiment, the above-mentioned reporter recombinant expression vector further comprises a marker gene expressing uracil.
According to a third aspect of the present invention, there is provided a method for regulating genome rearrangement based on unnatural amino acid, the method comprising:
transferring the recombinant expression vector of the second aspect into yeast comprising LoxP recombination sites; allowing the tool-set recombinant expression vector to express a tool-set comprising an unnatural aminoacyl-tRNA synthetase and an acting tRNA thereof, the anticodon of the tRNA being paired with a stop codon set in frame with the Cre recombinase encoding gene; in the presence of unnatural amino acid, reading and translating a stop codon in an reading frame of a Cre recombinase encoding gene into unnatural amino to generate a full-length functional Cre recombinase; under the action of the functional Cre recombinase, genomic rearrangement of the LoxP recombination site occurs.
In a preferred embodiment, the unnatural amino acid is O-methyl-L-tyrosine, the unnatural aminoacyl-tRNA synthetase is a methoxytyrosyl-tRNA synthetase, the stop codon is the codon encoding leucine 14 at the N-terminus of the Cre recombinase, the stop codon is TAG, and the tRNA is a tRNA with an anticodon of CUA.
In a preferred embodiment, the above method further comprises: the recombination efficiency is regulated by controlling the concentration of the unnatural amino acid so as to regulate the degree of synthesis type chromosome rearrangement regulation.
The invention provides a method for controlling synthetic genome rearrangement by site-directed introduction of unnatural amino acids. According to the invention, on the basis of the traditional method, the EBD domain combined with estradiol in the Cre-EBD fusion protein is deleted, so that the influence of reduced activity of Cre recombinase caused by protein fusion is eliminated. In addition, the function of Cre recombinase after EBD domain removal is no longer dependent on the hormone estradiol, thereby eliminating safety risks for experimenters. By combining with a codon expansion technology, the unnatural amino acid is introduced into a specific site of the Cre recombinase through an efficient orthogonal unnatural amino acid coding tool, and under the condition that the introduction of the unnatural amino acid does not influence protein folding and functions, the expression of the full-length Cre recombinase is accurately controlled by adding an unnatural amino acid substrate, so that the regulation and control of a genome rearrangement process are realized. The regulation and control tool has high orthogonality, so that the functional leakage of Cre recombinase is prevented, and the method has strong control force and accuracy. Furthermore, the efficiency of occurrence of genome rearrangement has been demonstrated to be regulated by the addition of different concentrations of unnatural amino acids, embodying the flexibility of the invention.
In sum, on the basis of safety, the invention well solves the problem of Cre recombinase function leakage, can effectively ensure the stability of genome, and can regulate the occurrence rate of genome rearrangement according to actual requirements. The invention greatly releases the potential of genome rearrangement in synthetic organisms, screens and obtains the synthetic saccharomyces cerevisiae with novel genotype, and is applied to a plurality of application scenes such as high-yield bacteria and environment-tolerant bacteria related to actual demands.
Drawings
FIG. 1 is a schematic diagram showing the structures of the report gene recombinant expression vectors pLH_Scr18 and pLH_Scr19 in the examples of the present invention;
FIG. 2 is a diagram showing the result of electrophoresis detection of a strain carrying a genome rearrangement control system according to an embodiment of the present invention;
FIG. 3 is a graph showing the comparison result of the genome rearrangement control method according to the embodiment of the present invention with the conventional genome rearrangement control method;
FIG. 4 is a graph showing the results of optimizing the genome rearrangement control system according to the embodiment of the present invention.
Detailed Description
The invention will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present invention. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.
Aiming at the defects of the SCRaMble system regulation and control method in the prior art, including the problems of function leakage, low recombination efficiency, estradiol toxicity and the like, the invention provides a brand new technology for coping with the defects, namely a novel genome rearrangement regulation and control system and method based on unnatural amino acid.
In one embodiment of the invention, the EBD domain of the Cre-EBD fusion protein that binds to estradiol is deleted by engineering the pSCW11-Cre-EBD plasmid in the conventional method, thereby minimizing the effect of protein fusion that results in a decrease in Cre recombinase activity. In addition, the invention utilizes a codon expansion technology to introduce unnatural amino acid into a specific site of the Cre recombinase, for example, a stop codon TAG is added in the middle of a coding frame of the Cre recombinase, so that the unnatural amino acid can be translated, and the genome rearrangement process can be regulated and controlled by controlling the Cre recombinase expressing the full length under the condition that the introduction of the unnatural amino acid does not influence the folding and the functions of the protein.
In one embodiment of the invention, expression of a protein carrying an unnatural amino acid by a codon extension technique requires the introduction of an orthogonal unnatural amino acid coding tool, which can be a methoxytyrosyl-tRNA synthetase/leucine tRNA orthogonal pair (LeuOmeRS/tRNA) CUA Where CUA represents an anticodon on the tRNA, which is complementary to the stop codon UAG), the coding tool is also referred to herein as "tool pairing". LeuOmeRS can specifically recognize the unnatural amino acid O-methyl-L-tyrosine (OmeY) in the presence of an unnatural amino acid substrate and specifically link it to a corresponding tRNA CUA The activated tRNA recognizes and reads through the stop codon, thereby generating full-length Cre recombinase, which interacts with LoxP recombination sites to undergo genomic recombination. When OmeY is not added, only inactivated truncated Cre recombinase is expressed in the cells and cannot interact with LoxP recombination sites to undergo genomic recombination. In addition, ome is a common unnatural amino acid, and toxicology information is recorded temporarily, so that the compound is safer for experimenters than estradiol. More importantly, the technology of the invention can also adjust the recombination efficiency by controlling the concentration of OmeY, and the expression efficiency of Cre recombinase is found to be positively correlated with the concentration of a non-natural amino acid substrate, so that the degree of SCRaMble can be accurately regulated.
On the basis of no toxicity and high efficiency, the invention well solves the problem of functional leakage of Cre recombinase, can effectively ensure the stability of genome, and can regulate the occurrence rate of genome rearrangement according to actual demands.
In one embodiment of the invention, a non-natural amino acid based genome rearrangement control system comprises: a tool pair for coding an unnatural amino acid, the tool pair comprising an unnatural aminoacyl-tRNA synthetase and an acting tRNA thereof, where the anticodon of the tRNA is paired with a stop codon set in frame of a Cre recombinase-encoding gene, where the stop codon is translated into the unnatural amino acid by the presence of the unnatural amino acid and the action of the tool pair; and a regulation means for controlling the expression of Cre recombinase, the regulation means comprising a Cre recombinase encoding gene comprising in its reading frame at least one set stop codon, which serves as an insertion site for an unnatural amino acid, producing a full-length functional Cre recombinase upon reading through the stop codon, and producing a truncated inactive Cre recombinase upon failing to read through the stop codon, said functional Cre recombinase being capable of acting on the recombination site LoxP to thereby cause genome rearrangement.
In the embodiment of the present invention, the unnatural amino acid is other than 20 natural amino acids, and the amino acids may be derivatives of natural amino acids, such as phenylalanine derivatives, tyrosine derivatives, glutamine derivatives, alanine derivatives, cysteine derivatives, serine derivatives, lysine derivatives, and the like. In one embodiment of the invention, the unnatural amino acid is O-methyl-L-tyrosine, and accordingly, the unnatural aminoacyl-tRNA synthetase is a methoxytyrosyl-tRNA synthetase.
The stop codon in the Cre recombinase-encoding gene may be a manually introduced stop codon, which may be introduced at any position of the encoding gene as long as the folding and function of the protein are not affected. In one embodiment of the invention, the stop codon is the codon encoding leucine 14 at the N-terminus of the Cre recombinase replaced with the stop codon TAG, and correspondingly the tRNA is the tRNA with the anticodon CUA.
In one embodiment of the invention, a recombinant expression vector for producing a genomic rearrangement control system of the invention comprises: a tool-paired recombinant expression vector comprising a vector backbone and a coding element of the tool-paired of the invention; and, a regulatory tool recombinant expression vector comprising a vector backbone and the coding elements of the regulatory tool of the invention.
In one embodiment of the invention, the tool-set recombinant expression vector further comprises a marker gene for leucine expression, the yeast transformed with the tool-set recombinant expression vector being capable of being cultured in a medium lacking leucine; the recombinant expression vector for regulatory means further comprises a marker gene for expressing histidine, and the yeast transformed with the recombinant expression vector for regulatory means can be cultured in a medium lacking histidine.
In order to verify the effect of the SCRaMbLE of the present invention, in one embodiment of the present invention, the recombinant expression vector further includes a reporter gene recombinant expression vector including a vector backbone and a reporter gene and a terminator carrying LoxP recombination sites at the head and tail before the reporter gene, and when rearrangement occurs, the terminator is deleted and the reporter gene is expressed normally. As shown in FIG. 1, in one embodiment of the present invention, the reporter recombinant expression vector includes reporter vectors pLH_Scr18 and pLH_Scr19, wherein the triangle structure is LoxP recombination site. When SCRaMble occurs, the sequence in the middle of two loxP recombination sites in the same direction is deleted, and the fluorescence of the bacterial cells increases. The positive control pLH_Scr19, without stop codon before GFP sequence, can be used as a positive control for 100% fluorescence. In one embodiment of the invention, the reporter recombinant expression vector further comprises a marker gene for uracil expression, and the yeast transformed with the reporter recombinant expression vector is capable of being cultured in a medium lacking uracil.
In one embodiment of the invention, a method for regulating genome rearrangement based on unnatural amino acid, the method comprising: transferring the recombinant expression vector (at least comprising a tool pair recombinant expression vector and a regulatory tool recombinant expression vector) of the invention into yeast comprising a LoxP recombination site; allowing the tool-set recombinant expression vector to express a tool-set comprising an unnatural aminoacyl-tRNA synthetase and an acting tRNA thereof, the anticodon of the tRNA being paired with a stop codon set in frame with the Cre recombinase encoding gene; in the presence of unnatural amino acid, reading and translating a stop codon in an reading frame of a Cre recombinase encoding gene into unnatural amino to generate a full-length functional Cre recombinase; under the action of the functional Cre recombinase, genomic rearrangement of the LoxP recombination site occurs.
The invention can precisely regulate and control the expression of Cre recombinase in the genome rearrangement process at the translation level, thereby preventing the function leakage of the enzyme. In addition, the expression of the aminoacyl tRNA synthetase gene in the coding tool can be controlled at the transcription level, so that a control system for jointly controlling genome rearrangement at the translation and transcription levels is realized, and the function leakage is further reduced. In addition, unnatural amino acids carrying photoprotection groups can be introduced at the active residue (e.g., lysine, cysteine) position of the Cre recombinase, thereby precisely modulating the activity of the Cre recombinase by illumination at specific wavelengths.
The method of non-natural amino acid regulated genomic rearrangement can also regulate gene switch expression, turning specific or random genes on or off by genomic rearrangement. In addition, the invention can also be used for directed evolution of strains to obtain strains with novel functions or enhanced specific functions.
The following detailed description of the present invention is provided by way of example only, and should not be construed as limiting the scope of the invention.
Example 1
This example describes a novel genome rearrangement control method based on unnatural amino acids, comprising: checking the effectiveness of tool pairing, establishing a novel genome rearrangement control system based on unnatural amino acid, obtaining a strain carrying the control system, determining the efficiency of the novel genome rearrangement control system based on the novel genome rearrangement control step of unnatural amino acid, and optimizing the novel genome rearrangement control system.
(1) Checking the validity of tool pairs
The availability of tool pairing was verified by using GFP fluorescent protein containing a stop codon as a reporter gene, as shown in FIG. 1, as a pLH_Scr18 reporter vector, and co-transferring the reporter vector with the tool pairing into yeast, and up-regulating the GFP expression level in cells after adding unnatural amino acid OmeY.
(2) Establishment of novel genome rearrangement regulation system based on unnatural amino acid
The regulatory system of this embodiment includes a pair of tools for unnatural amino acid encoding and a regulatory tool for controlling Cre recombinase expression. In this example, for the construction of the tool-paired vector, the universal pRS315 plasmid was used as the vector backbone, and the clone-insert tool was paired to give a plasmid called pXF231 carrying a leucine (Leu) -expressing marker gene; for the construction of a regulation and control tool vector, genome rearrangement (SCRaMble) is regulated by Cre recombinase, an EBD structural domain in Cre-EBD fusion protein is deleted through structural prediction, a codon of the N end of the Cre recombinase for encoding leucine 14 is selected to be replaced by a stop codon TAG for inserting unnatural amino acid, the constructed plasmid is named pXF, and the plasmid carries a marker gene for expressing histidine (His); as the reporter gene vectors, pLH_Scr18 and pLH_Scr19 were used, and as shown in FIG. 1, the plasmid carries a marker gene expressing uracil (Ura). The GFP sequence of pLH_Scr18 is preceded by a terminator with LoxP recombination sites at the head and tail, when rearrangement occurs, the terminator is deleted, GFP can be expressed normally, and the fluorescence of the thalli can be increased. The plh_scr19 had no terminator before GFP sequence, and the positive control of the reaction and the normalization for fluorescence value.
(3) Acquisition of strains carrying regulatory systems
Co-transferring pLH_Scr18, pXF, 238, pXF into yeast by yeast transformation technique, and applying to yeast selection medium SC -His/Leu/Ura (SC -3 ) On the above, the culture was carried out at 30℃for 48 hours to obtain a monoclonal antibody as an experimental group. Yeasts into which pLH_Scr19, pRS413 (blank vector) and pRS415 (blank vector) were transferred were used as positive controls for 100% GFP values. Strains of the Cre-EBD system (traditional regulation method) containing the galactose operon were used as controls for the efficiency of novel genome rearrangement regulation systems based on unnatural amino acids.
Yeast transformation is exemplified as follows:
(a) Wild type yeast monoclonal was selected and inoculated into 2mL of yeast enrichment medium YPD and drum-cultured at 30℃for 24 hours.
(b) The absorbance of the bacterial liquid is measured, a proper amount of bacterial liquid is inoculated into a triangle bottle containing 20mL YPD, the absorbance value of each milliliter of bacterial liquid is 0.1, and the bacterial liquid is cultured by rotating the shaking table 200 for each minute at 30 ℃ until the absorbance value of each milliliter of bacterial liquid is 0.6.
(c) And (5) centrifuging for 10 minutes at 2000 revolutions per minute, and removing the supernatant to collect bacterial liquid.
(d) 10mL ddH was added 2 O resuspended cells, 2000 revolutions per minute, centrifuged for 10 minutes, and the supernatant removed to collect the bacterial fluid.
(e) 10mL of 0.1M LioAc was added to resuspend the cells, and the cells were centrifuged at 2000 rpm for 10 minutes to collect the supernatant.
(f) 10mL of 0.1M LioAc was added to resuspend cells and competent cells were obtained, which were all the way to ice.
(g) Conversion reaction tubes were prepared, each containing the components of table 1 below:
TABLE 1
| Component (A) | Dosage of |
| Target DNA | X microliters (about 200 ng) |
| ddH 2 O | 22-X microliters |
| ssDNA | 25 microliters |
| 1M LioAc | 41 microliters |
| 50％PEG3350 | 312 microlitres |
(h) Mix well with shaking, add 100 μl of competent cells carefully to the uppermost layer.
(i) Mixing was reversed, and incubated at 30℃for 30 minutes.
(j) 50 microliters of DMSO was added and mixed quickly upside down.
(k) Heat shock was conducted at 42℃for 15 minutes and placed on ice for 1 minute.
(l) 8000 revolutions per minute Zhong Lixin seconds, the supernatant was removed.
(m) use 400. Mu.l of 5mM CaCl 2 The cells were resuspended and incubated at room temperature for 10 minutes.
(n) applying 50 microliters of the bacterial liquid to SC -3 On the medium, the culture was performed for 48 hours.
(o) obtaining the culture medium with the monoclonal is needed to carry out subsequent experiments in time, otherwise, the culture medium is preserved at 4 ℃.
(p) a method of screening for a strain containing a plasmid of interest: the monoclonal to be tested was picked up and dissolved in 50. Mu.l ddH 2 In O, 25. Mu.l of the bacterial liquid was dissolved in 25. Mu.l of 40mM NaOH, and the mixture was lysed at 99℃for 15 minutes, and allowed to stand at 4℃for 1 hour. 2.5 microliters of supernatant (note not to aspirate to pellet) was taken for colony PCR.
(q) colony PCR
The configuration reaction system is shown in the following table 2:
TABLE 2
| Component (A) | Dosage of |
| ddH 2 O | 2.25 microliters |
| Supernatant after cell lysis | 2.5 microliters |
| 10 mu M primer | 1.5 microliters |
| Gotaq Green Master Mix 2X | 6.25 microliters |
The cycle temperature settings are shown in table 3 below:
TABLE 3 Table 3
The result of the electrophoresis detection of the strain carrying the genome rearrangement control system is shown in FIG. 2, wherein the rightmost side is the molecular marker (1 kb DNA Ladder). If the colony PCR (Colony PCR) band is of the correct size, the strain is considered to contain the target plasmid, and can be selected for subsequent culture experiments. All colony PCR band sizes in the left panel were the same as pXF plasmid PCR Positive Control (PC) band sizes. The 8 bacteria in the figure are the same as the pXF238 plasmid PCR Positive Control (PC) band in size, indicating that these 8 bacteria can be used in subsequent experiments.
(4) Novel genome rearrangement control based on unnatural amino acids
Preparing a proper amount of culture solution:
(a) Yeast selective media are shown in Table 4 below:
TABLE 4 Table 4
| Reagent name | Added into 500ml |
| Double concentration Yeast three-deficiency culture Medium (2 XSC) -3 ) | 250ml |
| 40% glucose solution | 25ml |
| ddH 2 O | 225ml |
(b) Unnatural amino acid broth, as shown in table 5 below:
TABLE 5
| Reagent name | Added into 500ml |
| Double concentration Yeast three-deficiency culture Medium (2 XSC) -3 ) | 250ml |
| 40% glucose solution | 25ml |
| 50mmol/L OmeY | 10ml |
| ddH 2 O | 215ml |
(c) The remaining 25. Mu.l of the bacterial liquid after colony PCR was inoculated into 2mL of a yeast selective culture liquid, and the culture was subjected to drum culture at 30℃for 24 to 48 hours to obtain an activated bacterial liquid.
(d) The method for treating the unnatural amino acid regulatory group comprises the following steps: and (3) taking a proper amount of activated experimental group bacterial liquid, preparing bacterial liquid with the concentration of 0.05 per milliliter bacterial liquid by using the activated experimental group bacterial liquid and 2mL of non-natural amino acid culture liquid, and carrying out drum culture at 30 ℃ for 48 hours to obtain the non-natural amino acid regulating bacterial liquid.
(e) Control group treatment method: and (3) taking a proper amount of activated experimental group bacterial liquid, preparing bacterial liquid with the concentration of 0.05 per milliliter of bacterial liquid by using the activated experimental group bacterial liquid and 2mL of yeast selective culture liquid, and carrying out drum culture at 30 ℃ for 48 hours to obtain a control group bacterial liquid.
(5) Novel genome rearrangement regulatory system efficiency determination
The increase in fluorescence of the cells can be indicative of the efficiency of the control system, and the ratio of the amount of fluorescence of the test yeasts to the amount of fluorescence of the positive control yeasts is used as the relative fluorescence value.
And (3) collecting thalli: culturing for 48 hr, centrifuging at 3000 rpm for 5 min, removing supernatant, and re-suspending in 2mlddH 2 O.
Measurement preparation: selecting a specific 96-well plate for measuring fluorescence, selecting an A1 well, and driving into 200 microliters of ddH 2 O is used for measuring the background noise, and 200 microliters of bacteria liquid to be tested is injected into the residual hole.
The bacterial liquid was subjected to fluorescence measurement using a BioTek SYNERGY H1 microplate reader as an example: entering a Gene53.05 program, selecting a new scheme, selecting detection in operation, popping up a detection method popup window, selecting 'light absorption' in detection method options in the popup window, selecting 'end point/dynamics' in detection type options, selecting 'monochromator' in optical element type options, and clicking for determination. The detection step window pops up, the wavelength option selects "1", and fills in "600" in the box, clicking on the determination. The window disappears, a blank area under the existing detection is selected at the description option, the detection is selected in operation, the fluorescence intensity is selected in the detection method option in the popup window, the endpoint/dynamics is selected in the detection type option, the monochromator is selected in the optical element type option, and the click determination is performed. And (3) popping up a detection step window, wherein the wavelength options are selected to be '1', the wavelength options are all defaulted, the detection height is clicked to be 'automatically adjusted', the pop-up window is clicked to be 'started to calibrate', after the board to be detected is put into the board to be detected, the detection step window is clicked to be determined, the detected optimal height is set to be the detection height, the window disappears, and the click is confirmed. The window disappears, the clicking confirms, at this moment the carrier plate stage can pop up, will await measuring the board and place on the carrier plate stage, and click and confirm, the machine can begin to survey fluorescence. After the fluorescence measurement is completed, a derived table is obtained.
Data analysis: and subtracting the absorbance value and the fluorescence value of the water in the hole A from the absorbance value and the fluorescence value of the obtained thalli respectively to obtain the ratio of the corrected fluorescence value to the absorbance value as the fluorescence value of the thalli in unit concentration. And comparing the unit concentration fluorescence value of the different experimental groups with the unit concentration fluorescence value of the positive control group to obtain the relative fluorescence value of the different experimental groups.
As shown in FIG. 3, the ratio of the fluorescence value of the unit cell to the fluorescence value of the positive control (pLH_Scr19) unit cell is shown on the ordinate. Compared with the traditional galactose manipulation and regulation method, the novel regulation method based on the unnatural amino acid has almost no leakage and is close to the fluorescence value of the thalli. In addition, the novel method provided by the invention has obviously improved effect after induction.
(6) Novel genome rearrangement regulatory system optimization
Finding the optimal concentration of the unnatural amino acid OmeY, wherein the concentration of OmeY is too low, so that the regulation effect is not obvious, and the bacterial growth can be inhibited when the concentration is too high.
Gradient unnatural amino acid cultures (0.5 to 10 fold concentration) were prepared as shown in Table 6 below:
TABLE 6
The remaining 25. Mu.l of the bacterial liquid after colony PCR was inoculated into 2mL of a yeast selective culture liquid, and the culture was subjected to drum culture at 30℃for 24 to 48 hours to obtain an activated bacterial liquid.
And (3) taking a proper amount of activated experimental group bacterial liquid, culturing with 2mL of gradient unnatural amino acid to prepare bacterial liquid with the concentration of 0.05 of absorbance value of each milliliter of bacterial liquid, and carrying out drum culture at 30 ℃ for 48 hours to obtain the gradient unnatural amino acid regulating bacterial liquid.
The cells were collected and prepared in the same manner as described above, and the bacterial liquid was subjected to fluorescence measurement.
Data analysis: and subtracting the absorbance value and GFP value of water from the absorbance value and fluorescence value of the obtained thalli respectively to obtain the ratio of the corrected fluorescence value and absorbance value as the fluorescence value of the thalli in unit concentration. And comparing the fluorescence value of the unit concentration of the experimental group cultured by the gradient unnatural amino acid culture solution with the fluorescence value of the unit concentration of the positive control group to obtain the relative fluorescence values of different experimental groups.
The optimized result of the genome rearrangement control system is shown in fig. 4, and fig. A shows the growth condition of different bacteria after the culture of the gradient unnatural amino acid culture solution for 48 hours, wherein different concentrations in the abscissa refer to the concentration of unnatural amino acid in the culture solution, the 1-time concentration is 1mmol/L, and the ordinate indicates the concentration of bacteria. Panel B shows the amounts of the relative fluorescence values of different cells, the ordinate being the ratio of the fluorescence value of the cell units to the fluorescence value of the positive control (pLH_Scr19) cell units, the ratio reflecting the rearrangement efficiency. It can be seen that the growth of the bacterial cells is not significantly inhibited when the concentration of the unnatural amino acid in the culture solution is increased; at lower concentrations of unnatural amino acids (ome), the regulatory tools of the present method have demonstrated significant effects, and with increasing concentrations of ome, the rearrangement effect is significantly enhanced.
Claims (8)
1. A non-natural amino acid based genome rearrangement control system, the system comprising:
a tool pair for coding an unnatural amino acid, the tool pair comprising an unnatural aminoacyl-tRNA synthetase and an acting tRNA thereof, where the anticodon of the tRNA is paired with a stop codon set in frame of a Cre recombinase-encoding gene, where the stop codon is translated into the unnatural amino acid by the presence of the unnatural amino acid and the action of the tool pair; and
a regulation means for controlling Cre recombinase expression, the regulation means comprising a Cre recombinase encoding gene comprising in its reading frame at least one set stop codon for an insertion site of an unnatural amino acid, producing a full length functional Cre recombinase upon reading through the stop codon and a truncated inactive Cre recombinase upon failing to read through the stop codon, said functional Cre recombinase being capable of acting on the recombination site LoxP to thereby cause genome rearrangement;
the unnatural amino acid is O-methyl-L-tyrosine, and the unnatural aminoacyl-tRNA synthetase is a methoxytyrosyl-tRNA synthetase;
the stop codon is the substitution of the codon encoding leucine at position 14 at the N end of the Cre recombinase with the stop codon TAG, and the tRNA is the tRNA with the anticodon CUA.
2. A recombinant expression vector for producing the genomic rearrangement control system of claim 1, the recombinant expression vector comprising:
a tool-paired recombinant expression vector comprising a vector backbone and coding elements of said tool-paired; and
a regulatory tool recombinant expression vector comprising a vector backbone and coding elements of said regulatory tool.
3. The recombinant expression vector of claim 2, wherein the tool pair recombinant expression vector further comprises a marker gene that expresses leucine; the recombinant expression vector of the regulation tool also comprises a marker gene for expressing histidine.
4. The recombinant expression vector according to claim 2, further comprising a reporter recombinant expression vector comprising a vector backbone and a reporter gene and a terminator located before the reporter gene, the terminator carrying LoxP recombination sites end to end, wherein when rearrangement occurs, the terminator is deleted and the reporter gene is expressed normally.
5. The recombinant expression vector of claim 4, wherein the reporter gene is a GFP gene.
6. The recombinant expression vector of claim 4, wherein the reporter recombinant expression vector further comprises a marker gene that expresses uracil.
7. A method for regulating genome rearrangement based on unnatural amino acids, comprising:
transferring the recombinant expression vector of any one of claims 2 to 6 into yeast comprising a LoxP recombination site; allowing the tool-set recombinant expression vector to express a tool-set comprising an unnatural aminoacyl-tRNA synthetase and an acting tRNA thereof, the anticodon of the tRNA being paired with a stop codon set in frame with the Cre recombinase encoding gene; in the presence of unnatural amino acid, reading and translating a stop codon in an reading frame of a Cre recombinase encoding gene into unnatural amino to generate a full-length functional Cre recombinase; under the action of the functional Cre recombinase, genome rearrangement occurs at the LoxP recombination site; the unnatural amino acid is O-methyl-L-tyrosine, the unnatural aminoacyl-tRNA synthetase is methoxytyrosyl-tRNA synthetase, the stop codon is the codon encoding leucine 14 at the N-terminal end of the Cre recombinase, the stop codon is TAG, and the tRNA is the tRNA with the anticodon being CUA.
8. The method of claim 7, further comprising: the recombination efficiency is regulated by controlling the concentration of the unnatural amino acid so as to regulate the degree of synthesis type chromosome rearrangement regulation.
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