CN116286713A - High activity Tn5 transposase mutant - Google Patents
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Abstract
The invention discloses a high-activity Tn5 transposase mutant, which is obtained by mutating the site of the amino acid sequence of wild Tn5 transposase shown as SEQ ID No.3 to obtain a mutant of five site mutation of R26W, E54K, R62P, D156K, L372P, wherein the mutant is named Mut transposase, and the sequence of the mutant is shown as SEQ ID No. 4. Compared with wild type Tn5 transposase, the high-activity Tn5 transposase mutant has obviously improved activity.
Description
The application is a divisional application with the application number of 202210500786.8 and the application date of 2022, 5 and 10 days, and the invention name of a method for rapidly identifying Tn5 transposase activity.
Technical Field
The invention relates to a high-activity Tn5 transposase mutant, and belongs to the technical field of bioengineering.
Background
Tn5 transposase has now been applied as an important tool to high throughput sequencing technology, but the activity assay of transposase limits high throughput optimized screening and stable production of enzymes. The current activity assays for transposases mainly include three types: the first is based on the principle that transposase mediates insertion of reporter genes (resistance genes or toxic proteins) into the genome, and finally, the relative activity of transposase is indirectly determined through plate monoclonal counting, and the larger the difference between the cloning numbers of an experimental group and a control group is, the stronger the activity of transposase is. Although the method comprehensively considers two processes of shearing and pasting during transposition, the final monoclonal quantity, namely the determination of the enzyme activity, is influenced by the conversion rate and the cell state, and the determination result has larger error. In addition, the method is difficult to realize flux screening test due to large workload and complicated steps.
The second method relies on transposase-mediated dissociation of the quenching group from the fluorescent group, and determines the enzyme activity by the relative intensity of the fluorescent signal. Compared with the first method, the method can greatly reduce measurement errors and obtain enzyme activity results with better repeatability, but specific reagents and instruments are needed in the measurement process. And because the activity is tested in vitro, the expressed transposase needs to be purified, and the method is not suitable for screening large-scale mutant libraries and enzyme activity testing.
The third method directly compares the quality of library construction of different transposases, and indicates the intensity of enzyme activity according to the coverage of the library; or evaluating the activity of the novel transposase mutant based on the concentration of the PCR product of the adapter primer. The activity measured by the method can be directly related to downstream application, but the time and material cost for measuring the enzyme activity are extremely high, and particularly, the cost performance is relatively low when a large amount of screening work is carried out. Moreover, the library-building reaction requires higher purity enzymes, and the purification process is more demanding, and is also not suitable for high throughput screening.
Therefore, it is urgent to develop a rapid and accurate relative quantitative method of enzyme activity.
Disclosure of Invention
The invention provides a high-activity Tn5 transposase mutant, which is obtained by mutating the site of the amino acid sequence of wild Tn5 transposase shown as SEQ ID No.3 to obtain a mutant of five site mutation of R26W, E54K, R62P, D156K, L372P, wherein the mutant is named Mut transposase, and the sequence of the mutant is shown as SEQ ID No. 4.
The invention adopts the technical scheme that: a method for rapidly identifying Tn5 transposase activity, comprising the steps of:
(1) Constructing a reporter gene expression plasmid and a Tn5 transposase expression plasmid, wherein the reporter gene expression plasmid is a fluorescent protein expression plasmid, the reporter gene expression plasmid replicon is an escherichia coli replicon compatible with the pBR322 replicon, a promoter of the reporter gene expression plasmid is an inducible promoter, and sequences specifically recognized by the Tn5 transposase are added at two sides of the promoter;
(2) The reporter gene expression plasmid and the Tn5 transposase expression plasmid are co-transfected into an expression cell to construct a double-plasmid reporting system;
(3) Inducing Tn5 transposase expression;
(4) Inducing reporter gene expression;
(5) Detecting the level of reporter gene expression, and determining the relative activity of the transposase.
Preferably, the fluorescent protein in the reporter gene expression plasmid is red, green or blue fluorescent protein; the reporter gene expression plasmid replicon is a p15A replicon; the reporter gene expression plasmid promoter is P lac 、P BAD Or P tet The sequence specifically recognized by Tn5 transposase is IE, OE or ME.
Preferably, the Tn5 transposase expression plasmid is a pET series plasmid expressing Tn5 transposase.
Preferably, the Tn5 transposase expression plasmid is a pET21b (+) plasmid.
Preferably, the expression cell is an E.coli cell.
Preferably, the expression cell is BL21 (DE 3), rosetta (DE 3) or other E.coli cells containing T7 RNA polymerase.
Preferably, the conditions for inducing expression of Tn5 transposase are 0.5-5 mM IPTG,37℃and 250rpm for 4-5 hours, and Mg is added 2+ As an activator.
Preferably, the induction condition of the reporter gene expression is that after Tn5 transposase induction for 1-1.5 hours, the corresponding inducer is added according to the promoter type, and the culture is carried out for 3-4 hours at 37 ℃ and 250 rpm.
Preferably, the method for detecting the expression level of the reporter gene is to test the fluorescence intensity of fluorescent protein in cells or in a solution after ultrasonic disruption of cells, and compare the fluorescence intensity signal with the fluorescence intensity of a wild-type Tn5 transposase group of a control to obtain the relative fluorescence intensity of mutant Tn5 transposase.
Preferably, the method for detecting the expression level of the reporter gene comprises the following specific steps:
1) Measuring the absorbance of the cell culture solution at 600nm, and measuring the OD value to ensure that the number of cells taken in each experiment is consistent, and the final enzyme activity is comparable;
2) Taking a cell culture solution containing 3-5OD and centrifuging;
3) The cells were resuspended in 500 μl PBS and centrifuged;
4) Repeatedly washing the thalli;
5) Resuspend cells with PBS;
6) And taking a resuspended cell sample in the ELISA plate hole, and setting wavelength to test fluorescence intensity.
Preferably, the method for detecting the expression level of the reporter gene comprises the following specific steps:
1) Measuring the absorbance of the cell culture fluid at 600 nm;
2) Taking a cell culture solution containing 3-5OD and centrifuging;
3) The cells were resuspended in 500 μl PBS and centrifuged;
4) Repeatedly washing the thalli;
5) Resuspend cells with PBS;
6) Centrifuging after ultrasonic crushing, and collecting supernatant;
7) Taking a supernatant sample in the ELISA plate hole, and setting wavelength to test fluorescence intensity.
Preferably, the method further comprises the step (6): testing the abundance of the reporter gene expression plasmid, rechecking the transposase activity.
Preferably, the abundance of the test reporter gene expression plasmids is the number of reporter gene expression plasmids without promoter regions in the test cells, the same pair of primers (shown in figure 2) is adopted to form two PCR products of the size when two reporter plasmids with and without promoters are amplified, and the transposase activity is determined according to the ratio of small fragments to large fragments, and the larger ratio indicates the higher transposase activity.
The method for rapidly identifying the activity of the Tn5 transposase comprises the steps of firstly constructing a reporter gene expression plasmid, and when the reporter gene expression plasmid and the expression plasmid of the Tn5 transposase are transformed into escherichia coli cells together, the expressed Tn5 transposase recognizes specific sequences at two sides of a promoter at the front part of the reporter gene, and the promoter is stripped from the plasmid through transposition reaction. Reporter gene expression is then induced and Tn5 transposase activity is ultimately determined by testing the level of reporter gene expression and comparing it to a control level. The higher the transposase activity, the lower the reporter plasmid gene expression level. The invention can rapidly and sensitively judge the activity difference of the Tn5 transposase mutant or recombinant body relative to the wild Tn5 transposase by detecting the fluorescence intensity. The whole activity determination process only needs 30-60min, so that the enzyme activity determination time is greatly shortened and the test flux is greatly improved.
Compared with wild type Tn5 transposase, the high-activity Tn5 transposase mutant has obviously improved activity.
Drawings
FIG. 1 is a schematic diagram showing the principle of rapid activity determination of Tn5 transposase. pEGFP is a reporter plasmid engineered from pET28a (+); p (P) BAD Is an arabinose promoter, araC is a repressor expression gene, EGFP is an enhanced green fluorescent protein expression gene, and KanR is a kanamycin resistance screening gene; p15A ori is replicon.
FIG. 2 is a schematic diagram of the primers for PCR enzyme activity assay. ME is a transposase specific recognition sequence; primer F and primer R are a pair of PCR primers outside ME; the promoter is an inducible promoter.
FIG. 3 shows the results of a fluorescent intensity test for Tn5 transposase relative quantification. The control group is the fluorescence intensity of the intracellular fluorescent protein of the escherichia coli deleted of the transposase sequence in the transposase expression plasmid; tn5 group is the fluorescence intensity when the transposase expression plasmid expresses wild type Tn 5; mut panel is the fluorescence intensity of transposase expression plasmid expression mutant Tn5.
FIG. 4 shows the results of PCR electrophoresis of Tn5 transposase relative quantification. The control group is the PCR result of the report plasmid when the transposase sequence is deleted in the transposase expression plasmid; tn5 group is PCR result when the transposase expression plasmid expresses wild type Tn 5; mut panel is the PCR result of transposase expression plasmid expression mutant Tn5.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally performed under conventional conditions or conditions provided by the manufacturer. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
1. EGFP reporter plasmid construction
As shown in FIG. 1, the pBR322 replicon was replaced with the p15A replicon using the pET28a plasmid as a backbone; replacing the lactose operon with an arabinose operon; then, a green fluorescent protein gene (SEQ ID NO. 1) was inserted downstream of the arabinose promoter, and 3 sets of ME sequences (SEQ ID NO. 2) were ligated in tandem on both sides of the promoter.
2. Construction of expression strains
The Tn5 transposase expression plasmid and the reporter plasmid are co-transformed into an escherichia coli BL21 (DE 3) cell to construct a double-plasmid reporter system. Coli containing the double plasmid can be grown in the environment of kanamycin and carbenicillin double antibiotics. The Tn5 transposase expression plasmid was constructed as follows:
1) Synthesizing a wild type Tn5 gene sequence shown as SEQ ID No.3 (Tn 5 hereinafter) and a mutant type Tn5 gene sequence shown as SEQ ID No.4 (namely E54K and L372P mutant type Tn5 hereinafter Mut), connecting to a pET21b (+) vector after double digestion with NdeI/XhoI, transforming into DH5 alpha competent cells, coating a carboxybenzyl resistance plate, and culturing at 37 ℃ in an inverted manner overnight;
2) The monoclonal colony is picked up and inoculated into 5mL LB culture medium containing carbenicillin, and cultured at 37 ℃ and 250rpm for overnight;
3) By the company of the Saint Bio-on the Shanghai next
Plasmid is extracted by the Plasmid Mini Kit small amount extraction Kit and sequenced, and the correct sequence indicates that the transposase is completedAnd (5) constructing an expression plasmid.
3. Induction of transposase expression
E.coli BL21 (DE 3) monoclonal was selected, inoculated into 5mL LB liquid medium containing kanamycin and carbenicillin, cultured at 37℃and 250rpm for 3 hours, and then added with 0.5mM IPTG and MgCl with a final concentration of 20mM 2 Inducing and activating Tn5 transposase.
4. Induction of green fluorescent protein expression
After 1h of transposase expression, 0%, 0.005%, 0.02%, 0.08% or 0.32% L-arabinose was added to the medium, respectively, and the medium was incubated at 37℃for 4h at 250 rpm.
5. Collecting thallus and breaking cell wall by ultrasonic
The absorbance of each experimental group bacterial liquid at 600nm was measured, 5OD bacterial cells were taken out, washed 2 times with PBS, and centrifuged to discard the supernatant. The cells were resuspended in 500. Mu.L of PBS and the Tn5 transposase solution was obtained after 3min of sonication at 540Hz,4s/4 s.
6. Fluorescence value determination
100 mu L of bacterial liquid is added into a 96-well plate, 3 complex wells are arranged at each experimental group, the excitation light wavelength is 485nm, the emission light wavelength is 538nm, and fluorescent signals are measured under an enzyme-labeling instrument. As a result, as shown in FIG. 3, at each L-arabinose concentration, tn5 group was weaker in fluorescence than the control, mut group was weaker than Tn5 group, i.e., the mutated transposase (Mut) activity was significantly better than that of wild-type transposase Tn5. This method was shown to be directly useful for determining the difference in activity of mutated transposase from wild-type transposase.
7. Determination of the ratio of the two plasmids in the cell
And (3) taking 3 mu L of the residual solution in the step (6) as a template, and amplifying a reporter gene promoter region by using a primer F/primer R primer, wherein two strips of the size of the PCR product of the Mut group are obvious in the electrophoresis result of FIG. 4, and small fragments of the wild transposase group are not obvious. The gray scale scan results show that the brightness of the small fragments of the Mut group is improved by about 25%, namely, the relative enzyme activity of the Mut is improved by about 25%.
Primer sequences used:
primer F GAACCCCCCATGGTTAATTCCT
Primer R CATTGCCGTCACTGCGTCTTTT.
Claims (1)
1. A high-activity Tn5 transposase mutant is characterized in that the amino acid sequence of the mutant is shown as SEQ ID No. 4.
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| CN202210833604.9A CN116286713A (en) | 2022-05-10 | 2022-05-10 | High activity Tn5 transposase mutant |
| CN202210500786.8A CN114591993B (en) | 2022-05-10 | 2022-05-10 | Method for rapidly identifying activity of Tn5 transposase |
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| US20060294606A1 (en) * | 2004-05-18 | 2006-12-28 | Istefo Moisyadi | Tn5 transposase-mediated transgenesis |
| CN108265101A (en) * | 2016-12-30 | 2018-07-10 | 天津强微特生物科技有限公司 | A kind of Tn5 transposase enzyme activity determination methods of fast and stable |
| CN112899296A (en) * | 2019-12-04 | 2021-06-04 | 上海细胞治疗研究院 | Transposase screening report vector and preparation method and application thereof |
| CN112899252A (en) * | 2019-12-04 | 2021-06-04 | 上海细胞治疗研究院 | High-activity transposase and application thereof |
| CN114250266A (en) * | 2021-12-20 | 2022-03-29 | 南京诺唯赞生物科技股份有限公司 | Transposase activity determination method |
| CN114350693A (en) * | 2021-12-30 | 2022-04-15 | 苏州译酶生物科技有限公司 | Tn5 transposase and preparation method thereof |
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