CN116162639A - Mutant plasmid with controllable copy number and its application in phage-assisted evolution - Google Patents

Abstract

The invention discloses a plasmid with controllable copy number, which comprises a ColE1 replicon, and an operator is inserted downstream of the promoter of the ColE1 replicon. Also disclosed is a mutant plasmid with a controllable copy number, expressing enzymes that cause DNA damage and low-fidelity repair, the mutant plasmid includes a ColE1 replicon, and a cumate operator is inserted downstream of the promoter of the replicon. The application of the mutant plasmid in phage-assisted evolution and the phage-assisted evolution system are also disclosed. The present invention uses a phage-inducible promoter to control the copy number of the mutant plasmid, and at the same time uses a phage-inducible promoter to induce the expression of the mutant gene, which can ensure that the copy number of the mutant plasmid in the host bacteria not infected by the phage is low and the mutant gene The expression of the phage is inhibited to prevent meaningless background mutations, and after the phage infects the host bacteria, it can expand the copy number of the mutant plasmid and turn on the expression of the mutant gene, effectively improving the evolution efficiency of the phage genome.

Description

Mutant plasmid with controllable copy number and its application in phage-assisted evolution

technical field

The invention relates to a mutant plasmid with a controllable copy number and its application in phage-assisted evolution, as well as a corresponding phage-assisted evolution system, belonging to the technical field of gene evolution.

Background technique

Plasmid copy number refers to the number of plasmid vectors in the host bacteria, which is often determined according to the replicator of the plasmid vector itself (ori). There are many kinds of Ori, and different ori make the corresponding vectors have different copy numbers. Plasmids with different copy numbers have different application scenarios. Ori with high copy number, such as pMB1, pUC, RSF1030, etc., have 100-1000 copies in cells, and are often used for the preparation of a large number of DNA fragments, efficient non-toxic protein expression, etc.; ori with medium copy number, such as ColE1, pBR322, P15A , CloDF13, ColA, R6K, etc. have 15-50 copies in cells, and are often used for the expression of slightly toxic proteins and genes; low copy number ori such as pSC101, RK2, F1, P1, etc. have only 1-5 copies in cells A copy is usually used for the expression of highly toxic proteins and genes to reduce the impact of toxic proteins or genes on host cells, such as Cas protein, DNA damage protein, transposase, proteinase K, etc. However, in gene editing or gene mutagenesis, low-copy plasmids often have weak expression ability and affect the function of genes on the plasmid, especially in gene mutagenesis plasmids, low-efficiency mutagenesis plasmids will affect the speed of evolution. However, high-copy mutagenesis plasmids will cause serious background mutations to the evolutionary host bacteria before evolution, which will affect the quality of evolution. Therefore, it is very important to develop a method to control the copy number of plasmids during the evolution process.

Directed evolution is a biotechnology that has emerged in recent years. Using a laboratory-scale evolution system, the evolution results that take billions of years in nature can be obtained within a few months. It is a protein or RNA modification technology used. Phage-assisted strain evolution technology, also known as PACE technology (phage assisted continuous evolution), is a phage-based enzyme evolution technology developed by David R. Liu of Harvard University in 2011. The core of this technology is to couple the activity of the enzyme with the infection ability of the phage, that is, the stronger the activity of the enzyme, the higher the titer of the phage. The random mutagenesis method is used to continuously generate enzyme mutants during the evolution process, and the enzyme gene is integrated into the phage genome, so as to achieve the purpose of packaging a more active phage with higher activity enzyme genes carried on the genome. Finally, this method was used to screen out highly active enzyme variants. PACE has the advantages of simple operation, low cost and fast evolution, so it is widely used in various fields of enzyme evolution. However, the gene mutagenesis technology used by PACE is a high-copy plasmid, which largely causes background mutations and affects the operation and feasibility of PACE.

Contents of the invention

The purpose of the present invention is to provide a plasmid with a controllable copy number, the expression of which is affected by an external inducer or external pressure.

The technical scheme adopted in the present invention is: a plasmid with a controllable copy number, the plasmid comprises a ColE1 replicon, and an operon is inserted downstream of the RNA-II promoter of the ColE1 replicon, and the operon is a lactose operon, Arabinose operon, tetracycline operon or cumate operon.

Preferably, the sequence of the ColE1 replicon is shown in any one of SEQ ID NO: 1-4.

The invention also discloses an expression system with a controllable copy number, which includes the above-mentioned plasmid and Escherichia coli host bacteria, and the plasmid is transformed into the Escherichia coli host bacteria.

The invention also discloses a mutant plasmid with a controllable copy number, which expresses enzymes that cause DNA damage and low-fidelity repair, the mutant plasmid contains a ColE1 replicon, and is inserted downstream of the promoter of the ColE1 replicon cumate operon.

Preferably, the enzyme causing DNA damage and low-fidelity repair is low-fidelity DNA polymerase, DNA deaminase, DNA methylase, DNA demethylase, DNA glycosylase, DNA editing enzyme or DNA repair inhibitory enzymes.

Preferably, the gene promoter of the enzyme uses a phage-inducible promoter.

Preferably, the sequence of the mutant plasmid is shown in SEQ ID NO:5.

The invention also discloses the application of the above mutant plasmid in phage-assisted evolution.

The invention also discloses a phage-assisted evolution system with controllable plasmid copy number, including a host bacterium, a mutant plasmid, a gIII gene expression auxiliary plasmid and an M13 phage genome lacking the gIII gene, the mutant plasmid is the above-mentioned mutant plasmid.

Preferably, the host bacteria is Escherichia coli containing F plasmid.

The invention discloses a method for controlling the copy number of the plasmid. The method is to add a lactose operon, a tetracycline operon or an arabinose operon on the basis of the ColE1 replicon, so that the copy number of the plasmid is controlled by an inducer. In addition, the present invention uses a phage-induced promoter to regulate the expression of the ColE1 replicon, so that the copy number of the plasmid is at a low copy level when the phage is not infected, and the copy number is significantly increased to a high copy level after the phage infection. In the process of phage-assisted evolution, phage-inducible promoters are used to control the copy number of mutant plasmids, and phage-inducible promoters are used to induce the expression of mutant genes, so as to obtain more active enzymes that cause DNA damage and low-fidelity repair . This method can not only ensure that the copy number of the mutant plasmid in the host bacteria that is not infected by the phage is low and the expression of the mutant gene is suppressed during the evolution process, preventing meaningless background mutations, but also can prevent the host bacteria from being infected by the phage. The copy number of the mutant plasmid is expanded and the expression of the mutant gene is turned on, effectively improving the evolution efficiency of the phage genome.

Description of drawings

Figure 1 shows the IPTG-induced changes in the copy number of the plasmid pUC19-ColE1-LacO.

Figure 2 is tetracycline-induced changes in the copy number of plasmid pUC19-ColE1-tetO.

Figure 3 shows the copy number change of cumate-induced plasmid pUC19-ColE1-cuO.

Fig. 4 shows the copy number change of phage-induced plasmid pUC19-ColE1-PSPO.

Fig. 5 is a schematic diagram of the mutant plasmid induced by phage amplification.

Figure 6 shows the changes of mutant plasmids before and after phage infection.

Figure 7 shows the mutation rate of host genome and phage genome before and after phage infection.

Figure 8 is a comparison of mutation preferences.

Detailed ways

Below in conjunction with accompanying drawing, further illustrate the present invention through the embodiment, but not as limitation to the present invention. Specific materials and their sources used in embodiments of the invention are provided below. However, it should be understood that these are merely exemplary and not intended to limit the present invention, and materials with the same or similar type, model, quality, property or function as the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 Testing of the controllable CloE1 replication origin site.

The strength of the ColE1 replicon is regulated by its own two non-coding RNAs, RNA-positive (RNA-II) and RNA-negative (RNA-I). The expression of RNA-II can increase the plasmid copy number of ColE1 replicon, while RNA-I inhibits the plasmid copy number by inhibiting the activity of RNA-II. The expression of the two RNAs is mediated by their respective promoters, and the strength of the two promoters determines the ratio of RNA-I/RNA-II, which ultimately determines the copy number of the plasmid.

On the basis of this principle, the ColE1 replicon is modified by using the operon regulation model, so that the expression of RNA-I is controlled by an external inducer or external pressure (such as the control of phage infection).

1) The lactose operon LacO is inserted downstream of the RNA-II promoter, and the modified Ori sequence is shown in SEQ ID NO: 1, which is ColE1-LacO.

2) Insert the tetracycline operon tetO into the downstream of the RNA-II promoter, and the transformed Ori sequence is shown in SEQ ID NO: 2, which is ColE1-tetO.

3) The cumate operon cuO is inserted downstream of the RNA-II promoter, and the transformed Ori sequence is shown in SEQ ID NO: 3, which is ColE1-cuO.

4) Insert the phage-inducible promoter PSPO into the downstream of the RNA-II promoter, and the modified Ori sequence is shown in SEQ ID NO: 4, which is ColE1-PSPO.

The constructed sequence was constructed on the pUC19 plasmid and transformed into S1030 Escherichia coli.

qPCR SYBR Green Master Mix(No Rox)(翌圣生物)进行拷贝数定量检测。质粒拷贝数检测引物使用cgaaaagtgccacctgacgtc(SEQ ID No.7)和ccgcttacagacaagctgtgac(SEQID No.8)。对照引物使用gttagacgctgattacatgg(SEQ ID No.9)和gtcagtcaggaacagaccag(SEQ ID No.10)，扩增出大肠杆菌基因组的gapA基因的区域。定量结果见图1。The S1030/PUC19-ColE1-LacO strain was cultured in LB medium with 10 mg/L ampicillin until the OD600 value reached 0.3-0.5, and then 0, 0.01, 0.05, 0.1, 0.5, 1, and 2 mM final concentrations of IPTG were added, Induce overnight at 25°C. Centrifuge the thalli, use bacterial genomic DNA rapid extraction kit (Sangon Biotech, #B518225) to quickly extract bacterial DNA, use

qPCR SYBR Green Master Mix (No Rox) (Yisheng Biology) was used for quantitative detection of copy number. As primers for detecting plasmid copy number, cgaaaagtgccacctgacgtc (SEQ ID No. 7) and ccgcttacagacaagctgtgac (SEQ ID No. 8) were used. As control primers, gttagacgctgattacatgg (SEQ ID No. 9) and gtcagtcaggaacagaccag (SEQ ID No. 10) were used to amplify the region of the gapA gene of the E. coli genome. Quantitative results are shown in Figure 1.

qPCRSYBR Green Master Mix(No Rox)进行拷贝数定量检测。定量结果见图2。The S1030/PUC19-ColE1-tetO strain was cultured in LB medium with 10 mg/L ampicillin until the OD600 value reached 0.3-0.5, and then added 0, 0.01, 0.05, 0.1, 0.5, 1, and 2 mg/L final concentrations of Anhydrotetracycline, induced overnight at 25°C. Take the bacteria by centrifugation, and use the bacterial genome DNA rapid extraction kit to quickly extract the bacterial DNA, using

qPCRSYBR Green Master Mix (No Rox) for quantitative detection of copy number. Quantitative results are shown in Figure 2.

qPCR SYBR GreenMaster Mix(No Rox)进行拷贝数定量检测。定量结果见图3。The S1030/PUC19-ColE1-cuO strain was cultured in 10 mg/L ampicillin LB medium until the OD600 value reached 0.3-0.5, and then added 0, 0.1, 0.5, 1, 5, 10, 50 mg/L final concentrations of cumate, induced overnight at 25°C. Take the bacteria by centrifugation, and use the bacterial genome DNA rapid extraction kit to quickly extract the bacterial DNA, using

qPCR SYBR GreenMaster Mix (No Rox) for copy number quantification. Quantitative results are shown in Figure 3.

qPCRSYBR Green Master Mix(No Rox)进行拷贝数定量检测。定量结果见图4。S1030/PUC19-ColE1-PSPO strain was cultured in LB medium with 10 mg/L ampicillin until the OD600 value reached 0.3-0.5, and then added 0, 100, 500, 1000, 5000, 10000, 50000 CFU/ml of M13 phage , induced overnight at 25°C. Take the bacteria by centrifugation, and use the bacterial genome DNA rapid extraction kit to quickly extract the bacterial DNA, using

qPCRSYBR Green Master Mix (No Rox) for quantitative detection of copy number. Quantitative results are shown in Figure 4.

Example 2 Mutant plasmid test for phage-induced copy number changes and mutagenic gene expression.

A new type of PACE mutant plasmid was constructed using the phage-inducible promoter PSPO, which contains the following features:

1) The DNA mutation enzyme system uses low-fidelity DNA polymerase dnaQ926 and umuC, DNA deaminase aid and DNA editing enzyme abe, repair inhibitory enzyme ugi and emrR, etc. The combination of these enzymes is better, and one of them is used alone Enzymes are also available.

2) Using the PSPO promoter to express the DNA mutant enzyme system.

3) The replicon uses ColE1-PSPO.

4) See SEQ ID NO: 5 for the final mutated plasmid sequence, see Figure 5 for the schematic diagram of the vector, and see Figure 6 for the schematic diagram before and after M13 phage infection.

The mutagenic effects of the new PACE mutant plasmid and the traditional PACE mutant plasmid MP6 (addgene, #69669) were tested.

MP6 mutagenesis test: MP6 was transformed into JM109 (DE3) competent cells, cultured overnight at 37° C. on a solid plate containing 10 g/L glucose and 50 mg/L chloramphenicol. Pick a single clone and culture it in a liquid medium containing 10 g/L glucose and 50 mg/L chloramphenicol at 37° C. at 200 rpm until the OD600 reaches 0.4-0.5. Take 1 mL of the cultured strain, add 1 ml of M13 phage with a titer of 109 CFU/ml (the gIII gene is replaced by GFP, see SEQ ID NO: 6), incubate at room temperature for 30 min, and centrifuge at 5000 rpm for 5 min to obtain the precipitate. The strain was suspended in LB medium containing 0.2% arabinose, and cultured at 37° C. at 100 rpm for 3 h.

New mutation plasmid mutagenesis test. The new mutant plasmid was transformed into JM109 (DE3) competent medium, and cultured overnight at 37° C. on a solid plate containing 10 g/L glucose and 50 mg/L chloramphenicol. Pick a single clone and culture it in a liquid medium containing 10 g/L glucose and 50 mg/L chloramphenicol at 37° C. at 200 rpm until the OD600 reaches 0.4-0.5. Take 1mL of the cultured strain, add 1ml of M13 phage with a titer of 109CFU/ml (gIII gene is replaced by GFP), incubate at room temperature for 30min, and then centrifuge at 5000rpm for 5min to get the precipitate. Suspend the strains with fresh LB medium, culture at 37°C, 100rpm for 3h.

Use bacterial genomic DNA quick extraction kit to extract bacterial DNA rapidly, utilize the method for PCR to amplify, PCR primer uses primer pair gcaccaccggcaaactgc (SEQ ID No.11) and gcctttcagttcaatgcggttc (SEQID No.12) (amplification GFP region, representative mutation on the phage genome), the primer pair tggcgaggcgtggtcttcgtgg (SEQ ID No. 13) and cagcgtattcaggtgcgagttcg (SEQ ID No. 14) (amplifies the T7 RNAP region, representing a mutation on the host genome). The amplified fragments were verified by next-generation sequencing. The results are shown in Figures 7 and 8. The background mutagenesis rate of the mutagenesis plasmid designed by the present invention is lower, and the target mutagenesis rate is higher. speed.

Claims (10)

1. A plasmid with a controllable copy number, characterized in that the plasmid comprises a ColE1 replicon, and an operon is inserted downstream of the RNA-II promoter of the ColE1 replicon, and the operon is a lactose operon, an arabinose operator operon, tetracycline operon or cumate operon. 2. The plasmid with controllable copy number according to claim 1, characterized in that the sequence of the ColE1 replicon is as shown in any one of SEQ ID NO: 1-4. 3. An expression system with a controllable copy number, characterized in that it comprises the plasmid according to claim 1 or 2, and an Escherichia coli host bacterium, and the plasmid is transformed into the Escherichia coli host bacterium. 4. A mutant plasmid with a controllable copy number, said mutant plasmid expresses enzymes that cause DNA damage and low-fidelity repair, is characterized in that said mutant plasmid comprises a ColE1 replicon, and is inserted downstream of the promoter of the ColE1 replicon cumate operon. 5. The mutant plasmid according to claim 4, characterized in that the enzymes that cause DNA damage and low-fidelity repair are low-fidelity DNA polymerase, DNA deaminase, DNA methylase, DNA demethylase enzymes, DNA glycosylases, DNA editing enzymes, or DNA repair inhibitory enzymes. 6. The mutant plasmid according to claim 4, characterized in that the gene promoter of the enzyme uses a phage-inducible promoter. 7. The mutant plasmid according to any one of claims 4-6, characterized in that the sequence of the mutant plasmid is as shown in SEQ ID NO:5. 8. The application of the mutant plasmid according to any one of claims 4-7 in phage-assisted evolution. 9. A phage-assisted evolution system of controllable plasmid copy number, comprising a host bacterium, a mutant plasmid, a gIII gene expression helper plasmid and an M13 phage genome lacking the gIII gene, characterized in that said mutant plasmid is claimed The mutant plasmid described in any one of 4-7. 10. The phage-assisted evolution system according to claim 9, characterized in that the host bacterium is Escherichia coli containing F plasmid.

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