GB2591209A - High-Throughput screening method of lysine decarboxylase - Google Patents
High-Throughput screening method of lysine decarboxylase Download PDFInfo
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- GB2591209A GB2591209A GB2105916.7A GB202105916A GB2591209A GB 2591209 A GB2591209 A GB 2591209A GB 202105916 A GB202105916 A GB 202105916A GB 2591209 A GB2591209 A GB 2591209A
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Abstract
A high-throughput screening method for lysine decarboxylase includes the steps of: (I) introducing a plasmid containing a lysine decarboxylase gene into a lysine auxotrophic strain to obtain a recombinant bacterium; and (2) culturing the recombinant bacterium obtained in step (I) in a lysine-containing culture medium, and screening for lysine decarboxylase activity by measuring OD600. The invention couples the growth state of the lysine auxotrophic strain with the enzyme activity of the lysine decarboxylase, and does not need to detect consumption of substrates and generation of products. It may be used for the directed evolution of the lysine decarboxylase genes
Description
HIGH-THROUGHPUT SCREENING METHOD OF LYSINE
DECARBOXYLASE
TECHNICAL HELD
The present invention belongs to the field of biotechnics, and particularly relates to a high-throughput screening method of lysine decarboxylase.
BACKGROUND
Lysine decarboxylase is a biological enzyme that specifically decarboxylates L-lysine, which has the catalytic characteristics of high specificity and very high enzyme activity under suitable reaction conditions; but has the characteristics of narrow pH range and poor temperature stability. Therefore, developing a high-throughput screening method of lysine decarboxylase has practical significance to directed evolution of the lysine decarboxylase.
A CRISPR-Cas system is an immune defense system of bacteria, which is formed by evolution of the bacteria in a long-term process of resisting exogenous DNAs, and can degrade the invading exogenous DNAs (virus or phage, etc.), and widely exists in bacteria and archaea. A type II CRISPR-Cas9 system is widely used in gene editing at present. The system is mainly composed of three parts: a crRNA which targets a target sequence, a tracrRNA which can be paired with the crRNA, and a Cas9 protein which digests the target sequence. The CRISPR sequence transcripts the mature crRNA to be paired with the tracrRNA by complementary base, forming a partially double-stranded RNA structure, and then forming a complex with the Cas9 protein to cut off the exogenous DNA. Researchers integrated the crRNA and the tracrRNA into the same single strand, and designed a sgRNA (single guide RNA) with a length of about 20 bp. The sgRNA has two functions, i.e., the sgRNA can complement and pair with a target DNA sequence, and meanwhile, can guide the Cas9 protein to realize gene knockout. In recent years, the CRISPR-Cas9 technology has been widely used in genome editing and functional research of microorganisms, especially in the gene editing of bacteria, and a great progress has been made.
SUMMARY
Objective of the present invention: a technical problem to be solved by the present invention is to provide a high-throughput screening method of lysine decarboxylase aiming at
the deficiencies of the prior art.
In order to solve the foregoing technical problem, the present invention discloses a high-throughput screening method of lysine decarboxylase, including the following steps of: (1) introducing a plasmid ptrc-cadA containing lysine decarboxylase cadA gene into a lysine auxotrophic strain MG1655A1ysA to obtain a recombinant engineering bacterium MG1655AlysA/ptrc-cadA (referred to as MGAA); and (2) culturing the recombinant engineering bacterium obtained in step (1) in a lysine-containing culture medium, and screening expression oflysine decarboxylase by measuring 01360o.
In step (1), the lysine auxotrophic strain cannot grow normally in a culture medium without additional lysine.
In step (1), the lysine auxotrophic strain is constructed by knocking out a lysA gene in an E.coli MG1655. Preferably, the lysA gene in the MG1655 is knocked out by using a CRISPR-Cas9 editing technology.
In step (1), a nucleotide sequence of the lysine decarboxylase is shown as SEQ ID NO.1. In step (1), a nucleotide sequence of the plasmid ptrc-cadA containing lysine decarboxylase gene is shown as SEQ ID NO.5.
The plasmid ptrc-cadA containing lysine decarboxylase gene is constructed by digesting and then ligating cadA from a MG1655 by PCR replication with a vector plasmid ptrc (pTRC99a(Alac0)).
In primers for PCR replication, an upstream primer has a Nco I restriction site, and a downstream primer has a BamH I restriction site.
The vector plasmid ptrc is constructed by removing bases of a lac() part in the pTRC99a, wherein a nucleotide sequence of the vector plasmid ptrc is shown as SEQ ID NO.4.
In step (1), the recombinant engineering bacterium MG1655AlysA/ptrc-cadA cannot grow normally in a culture medium without additiona lysine; and is restricted to grow in the lysine-containing culture medium (high-activity decarboxylase will consume the lysine and hamper the growth of the recombinant engineering bacterium; while the growth is not affected in a culture medium without decarboxylase activity).
In step (2), the culturing in the lysine-containing culture medium is to pre-culture in a LB culture medium for 4 hours to 8 hours, and then to transfer to the lysine-containing culture medium for secondary culture for 10 hours to 14 hours. Preferably, the pre-culture lasts for 6 hours, and the secondary culture lasts for 12 hours.
In step (2), the culture medium is a composition of lysine and a M9 culture medium.
The concentration of the lysine in the culture medium is 0.1g/L to 2 g/L.
In step (2), a temperature of the pre-culture and culture is 34°C to 44°C, and preferably 37°C.
In step (2), the screening of lysine decarboxylase by measuring 0D600 is to couple a growth of MGAA with the enzyme activity of the lysine decarboxylase because the lysine decarboxylase has linear influence on the growth of the recombinant engineering bacteria MGAA under different enzyme activities; particularly, when the enzyme activity of the lysine decarboxylase is higher, the lysine decarboxylase will hamper the growth of the recombinant engineering bacterium MGAA, so that the ()Dot) is reduced, while when the enzyme activity of the lysine decarboxylase is lower, the lysine decarboxylase will not affect the growth of the recombinant engineering bacterium MGAA, so that the Moo will not be reduced.
In step (2), by measuring the 0D600 of different recombinant engineering bacteria in the same batch, one recombinant engineering bacterium with the lowest OD600 value is selected as the recombinant engineering bacterium capable of expressing the lysine decarboxylase with the highest enzyme activity, or several groups of recombinant engineering bacteria with lower 0D600 values are selected as the recombinant engineering bacteria capable of expressing the lysine decarboxylase with a higher enzyme activity. The recombinant engineering bacteria capable of expressing the lysine decarboxylase with high enzyme activity can be easily screened from a batch of the recombinant engineering bacteria expressing lysine decarboxylase with different enzyme activities by the method of the present invention.
Further, the present invention can culture the recombinant engineering bacteria expressing the lysine decarboxylase with high enzyme activity to obtain lysine decarboxylase with high enzyme activity.
Beneficial effects: compared with the prior art, the present invention has the following advantages: 1. The present invention screens the lysine decarboxylase based on the lysine auxotrophic strain MG1655A1ysA which is simple in construction and easy to realize.
2. The construction method of the plasmid ptrc-cadA expressing the lysine decarboxylase in the present invention is simple, and as the basis of cadA directed evolution, the screening steps are reduced.
3. The invention couples the growth state of the lysine auxotrophic strain with the enzyme activity of the lysine decarboxylase, does not need to detect consumption of substrates and generation of products, and is faster and more efficient, implements coupling of the growth state of the with the enzyme activity of the lysine decarboxylase, and has far-reaching significance to of the lysine decarboxylase.
BRIEF DESCRIPTION OF THE DRAWINGS
The advantages of the above-mentioned and/or other aspects of the present invention will become clearer by further explaining the present invention with reference to the accompanying drawings and specific embodiments.
FIG. 1 shows a gel electrophoresis for verifying construction of a lysine auxotrophic strain MG1655A1ysA, which is a gel electrophoresis of successful knockout of a lysA gene. FIG. 2 is a schematic diagram showing construction of a plasmid ptrc-cadA.
FIG. 3 is a diagram showing a growth of recombinant MG1655AlysAiptrc (without cadA) and MG1655AlysA/ptrc-cadA (MGAA with cadA) in an M9 medium added with lysine.
FIG. 4 is a diagram showing a linear relationship between an enzyme activity of lysine decarboxylase and a cell OD600.
DETAILED DESCRIPTION
The following description of the specific embodiments will further illustrate the present invention, but this is not a limitation to the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention, which shall all fall within the scope of the present invention as long as these modifications or improvements do not deviate from the basic idea of the present invention.
The experimental methods used in the following embodiments are conventional unless otherwise specified. The reagents and reagents used are commercially available unless otherwise specified. The technologies not mentioned in the embodiments are all conventional technologies in the field. in addition, the materials used such as MG1655 and pTrc99a are commercial products and can be purchased directly.
Embodiment 1: Construction of lysine auxotrophic strain MG1655AlysA A lysA gene of MG1655 was firstly knocked out by a CRISPR-Cas9 editing technology, wherein a knockout verification gel electrophoresis thereof was as shown in FIG. 1, a lysine auxotrophic strain MG1655(AlysA) was obtained.
Embodiment 2: Construction of plasmid ptrc-cadA (1) A whole genome of MG1655 was used as a template, and a coding sequence of lysine decarboxylase (cadA) on the MG1655 was amplified by conventional PCR.
An upstream primer had a Nco I restriction site and a nucleotide sequence thereof was shown as SEQ ID No.2, while a downstream primer had a BamH I restriction site and a nucleotide sequence thereof was shown as SEQ ID No.3: CATGccatggATATGAACGTTATTGCAATATTGAATCAC (SEQ ID No. 2); and CGCggatccTTATTTITTGCTTICTICTTTCAATACC (SEQ ID No. 3).
Reaction conditions were: 95°C for 2 minutes, 95°C for 20 seconds, 55°C for 20 seconds, 72°C for 165 seconds, and totally 30 cycles; and then 72°C for 5 minutes. The obtained sequence was electrophoresed by 1% agarose gel and corresponding fragments were recovered.
(2) The sequence obtained in step (1) and a vector pTrc99aAlac0 were digestedby Nco I and BamH I from Takara Company, and a digestion reaction system was: 2 gL of 10xbuffer H, 1 AL of Nco 1,1 gL of BamH I, 3 AL of gene fragment and ptrc vector, and 10 pi, of 1120. The -s -enzyme digestion system reacted at 37°C for 2 hours. Enzyme digestion products were ligated at 25°C for 3 hours, and a reaction system was: 1 pL of 10xLigase buffer, 1 pL of T4 DNA Ligase (Takara), 7 pl., gene fragment and 1 pt of vector.
(3) The ligation products obtained in step (2) were transformed into Transl -T1. A positive strain Trans 1 -T1-pTrc99a-rGCS was screened by PCR and then subjected to DNA sequencing to verify that the recombinant plasmid was constructed correctly.
(4) The positive strain was inoculated into a 5 mL LB/Amp liquid nutrient medium, wherein the LB/Amp liquid nutrient medium consisted of 10 g/L of peptone, 5 g/L of yeast powder and 5 g/L of sodium chloride, and then the positive strain was subjected to shaking culture overnight under the conditions of 37°C and 200 rpm. 12 hours later, the plasmid ptrc-cadA was extracted according to the operation instructions of Tiangen plasmid extraction kit. A construction diagram of the plasmid was as shown in FIG. 2.
Embodiment 3: Construction of recombinant MG1655(AlysA)/ptrc-cadA 2 pL of ptrc-cadA plasmid was transferred into a lysine auxotrophic strain MG1655AlysA by heat shock transformation, to obtain recombinant colibacillus MG1655(AlysA)Iptrc-cadA(MGAA).
Embodiment 4: Implementation of high-throughput screening of lysine decarboxylase Ingredients of M9 culture included: 10 g/L of NH4C1, 0.5 g/L of NaCl, 17.1 g/L of Na2HPO4.12H20, 3 g/L of 1CH2PO4, 0.12 g/L of MgSO4, and 1.1x10-2 g/L of CaC12.
Ingredients of LB culture medium included: 10 g/L of peptone, 5 g/L of yeast powder, and 5 g/L of NaCl.
A lysine auxotrophic strain MG1655(AlysA)/ptrc and a recombinant engineering bacterium MGAA were inoculated into a 5 in.L LB culture medium, pre-cultured at 37°C for 6 hours, then transferred to an M9 culture medium containing different concentrations of lysine, and cultured at 37°C for 12 hours, and then the growth of cell (0D600) was measured.
The results were as shown in FIG. 3: (1) a growth trend of the lysine auxotrophic strain MG1655(AlysA) in 0 g/L to 1 g/L of lysine was consistent with a concentration of the lysine; and (2) when the concentrations of the lysine in the culture medium were 0.5 g/L and 1 g/L, when the lysine decarboxylase expressed by cadA was active, the lysine in the culture medium was consumed, which inhibited the growth of the lysine auxotrophic strain and reduced the concentration of the OD600. Therefore, when qualitatively evolving the cadA, the enzyme activity of the mutated cadA can be determined by the above method. Because this method only needs to determine 013600, a microplate reader can be used for detection to realize high-throughput screening.
Embodiment 5: After random mutation was carried out on the lysine decarboxylase, MG1655(AlysA) was introduced to obtain recombinant engineering bacteria. A single colony was selected, inoculated into an M9 culture medium containing 1 g/L of lysine, and cultured at 37°C for 12 hours, and then 0D600 was measured. The recombinant engineering bacteria with OD600 of 0.2, 0.52, 0.8, 1.5, and 2 were taken respectively, and after being cultured alone, lysine decarboxylase was produced and an enzyme activity thereof was measured to obtain FIG. 4, which showed that there was a good linear relationship between the enzyme activity and the corresponding cell 013500, and the 013600 could be used to screen the lysine decarboxylase.
A definition of the enzyme activity of the lysine decarboxylase was: one enzyme activity unit, referring to an amount of enzyme that could transform 1 mmol of substrates in 10 minutes at 37°C and pH 7.0.
The present invention provides an idea and a process for the high-throughput screening method of lysine decarboxylase. There are many methods and ways to realize the technical solutions. The above is only the preferred embodiments of the present invention. It should be pointed out that those of ordinary skills in the art can make some improvements and embellishments without departing from the principle of the present invention, and these improvements and embellishments should also be regarded as falling with the scope of protection of the present invention. All the unspecified components in the embodiments can be realized by the prior art.
Claims (10)
- CLAIMS1. A high-throughput screening method of lysine decarboxylase, comprising the following steps of: (1) introducing a plasmid containing lysine decarboxylase gene into a lysine auxotrophic strain to obtain a recombinant engineering bacterium; and (2) culturing the recombinant engineering bacterium obtained in step (1) in a lysine-containing culture medium, and screening the recombinant engineering bacterium expressing lysine decarboxylase by measuring ODsoo.
- 2. The method according to claim 1, wherein in step (1), the lysine auxotrophic strain is constructed by knocking out a lys.,4 gene in an E.coli MG1655.
- 3. The method according to claim 1, wherein in step (1), a nucleotide sequence of the lysine decarboxylase is shown as SEQ ID NO.1.
- 4. The method according to claim 1, wherein a nucleotide sequence of the plasmid containing lysine decarboxylase gene is shown as SEQ ID NO.5.
- 5. The method according to claim 1, wherein the lysine decarboxylase gene was from DNA of MG1655 by PCR, and cloned into plasmid of ptrc to generate ptrc-cadA.
- 6. The method according to claim 5, wherein in primers for PCR replication, an upstream primer has a Nco I restriction site, and a downstream primer has a BamH I restriction site.
- 7. The method according to claim 5, wherein a nucleotide sequence of the vector plasmid ptrc is shown as SEQ ID NO.4.
- 8. The method according to claim 1, wherein in step (2), a concentration of the lysine in the lysine-containing culture medium is 0.1g/L to 2 g/L.
- 9. The method according to claim 1, wherein in step (2), the culturing is to pre-culture in a LB culture medium for 4 hours to 8 hours, and then to transfer to the lysine-containing culture medium for secondary culture for 10 hours to 14 hours.
- 10. The method according to claim 1, wherein in step (2), a temperature of the culturing is 34°C to 44°C.
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| Biotechnology & Bioprocess Engineering (2015); Vol 20, pp 439-446, "Directed Evolution and Mutagenesis of Lysine Decarboxylase...", Wang et al * |
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