Gene ANSB knockout mutant of citrobacter werkmanii and application thereof

Abstract

A gene ansB knockout mutant of Citrobacter werkmanii and an application thereof are provided. The gene ansB knockout mutant of the C. werkmanii is C. werkmanii with a gene ansB knocked out and a nucleotide sequence of the gene ansB is as shown in SEQ ID NO: 1. In the present invention, the acquired engineering bacteria with the gene ansB of the C. werkmanii knocked out are cultured in LB, TSB, NB and other media at 25° C. and 30° C., so that a biofilm formation capacity of the C. werkmanii on a polypropylene material is improved. Thus, the application scenarios and scopes of the C. werkmanii in heavy metal ion adsorption and construction of cellular protein synthesis micro-factories are broadened.

Claims

1. C. werkmanii ΔansB, wherein the C. werkmanii ΔansB has an accession No. GDMCC 61849.

2. A method for improving a biofilm formation capacity of C. werkmanii, wherein the biofilm formation capacity of the C. werkmanii is improved by knocking out a gene ansB of the C. werkmanii; the C. werkmanii is C. werkmanii GDFMZ BF-8 having an accession No. GDMCC 61858; and a nucleotide sequence of the gene ansB is as shown in SEQ ID NO: 1.

3. The method according to claim 2, wherein upstream and downstream homologous fragments of the gene ansB are amplified by using PCR, ligated with a plasmid pYG4 to construct a knockout vector pYG4-ansB, and then transformed with E. coli S17-1; the E. coli S17-1 carrying the knockout vector pYG4-ansB and the C. werkmanii are subjected to conjugational transfer to acquire a gene ansB knockout mutant of the C. werkmanii.

4. The method according to claim 3, wherein specific steps are as follows: (1) primer sequences: ansB-up-F: aaaagtgccacctgcagatctTTCGATATTTGGTGGGACTAAGTAGC (SEQ ID NO: 6); ansB-up-R: gccacctgcatcgaGTTATTTCTCCAGTTACTTGAATTTGC (SEQ ID NO: 7); ansB-down-F: aataacTCGATGCAGGTGGCTGCG (SEQ ID NO: 8); ansB-down-R: agtcatatgccgcggagatctCGGTCTGGGGCTACGTAGC (SEQ ID NO: 9); ansB-QJ-F: CGCTGGAAAACGATCGTAAAAC (SEQ ID NO: 10); and ansB-QJ-R: CAAGCCGTTCGAGTTCTTTATG (SEQ ID NO: 11); (2) upstream and downstream homologous sequences of the gene ansB are acquired by an amplification by taking an extracted C. werkmanii genome DNA as a template and taking the ansB-up-F, the ansB-up-R, the ansB-down-F, and the ansB-down-R as primers; (3) the plasmid pYG4 is subjected to single enzyme digestion with BglII and recovered by gel cutting; (4) the amplified upstream and downstream homologous fragments of the gene ansB are ligated with the plasmid pYG4 to construct the knockout vector pYG4-ansB, and then transformed with the E. coli S17-1 by heat shock; (5) the E. coli S17-1 carrying the knockout vector pYG4-ansB and the C. werkmanii are co-cultured to obtain a co-culture, and the co-culture is eluted, diluted and spread on a kanamycin- and rifampicin-resistant screening LB plate, and a gene ansB recombinant acquired by a one-time exchange is identified by using the ansB-QJ-F and the ansB-QJ-R as knockout identification primers; and (6) the gene ansB recombinant acquired by the one-time exchange is subjected to an amplification culture in an LB liquid medium, diluted and spread on an LB plate containing 5% sucrose by mass fraction, and a single clone is picked and identified with the knockout identification primers ansB-QJ-F and ansB-QJ-R to acquire the gene ansB knockout mutant of the C. werkmanii.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a PCR identification diagram of an ansB knockout strain ΔansB of C. werkmanii (lane 1: Marker III purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; lane 2: an upstream fragment of ansB; lane 3: a downstream fragment of ansB; and lane 4: knockout identified fragment); and

(2) FIG. 2A shows the biofilm formation of a wild strain BF-8 of and an ansB knockout strain ΔansB of C. werkmanii at 25° C. and under static culture; FIG. 2B shows the biofilm formation of a wild strain BF-8 of and an ansB knockout strain ΔansB of C. werkmanii at 30° C. and under static culture; FIG. 2C shows the biofilm formation of a wild strain BF-8 of and an ansB knockout strain ΔansB of C. werkmanii at 37° C. and under static culture; FIG. 2D shows the biofilm formation of a wild strain BF-8 of and an ansB knockout strain ΔansB of C. werkmanii at 25° C. and under shake culture at 120 rpm; FIG. 2E shows the biofilm formation of a wild strain BF-8 of and an ansB knockout strain ΔansB of C. werkmanii at 30° C. and under shake culture at 120 rpm; FIG. 2F shows the biofilm formation of a wild strain BF-8 of and an ansB knockout strain ΔansB of C. werkmanii at 37° C. and under shake culture at 120 rpm. Note: numbers above columns are average values.

DETAILED DESCRIPTION

(3) The following embodiments are intended to further illustrate the present invention, but not to limit the present invention.

(4) Wild C. werkmanii used in the following embodiments is C. werkmanii GDFMZ BF-8.

Embodiment 1

(5) I. Construction of ansB Knockout Vector

(6) An upstream homologous sequence (978 bp; its nucleotide sequence was as shown in SEQ ID NO: 2), a downstream sequence (795 bp; its nucleotide sequence was as shown in SEQ NO: 3) and a plasmid pYG4 sequence (5796 bp; its nucleotide sequence was as shown in SEQ ID NO: 4) of a gene ansB (1047 bp; its nucleotide sequence was as shown in SEQ ID NO: 1 and specifically was: ATGGAGTTTTTCAAGAAAACGGCACTTGCCGCACTGGTTATGGGTTTCAGCGGCGCG GCGCTTGCACTGCCAAACATCACTATTTTAGCAACCGGCGGGACCATTGCCGGCGGT GGTGATTCCGCGACAAAATCTAACTACACGGCAGGCAAGGTAGGCGTAGAGAATCT GGTTGAAGCCGTACCTCAGTTGAAAGACATCGCGGTTGTTAAAGGCGAGCAGGTGG TGAACATCGGCTCTCAGGATATGAATGACGACGTCTGGTTAACGCTGGCGAAAAAG ATTAACACCGAGTGTGATAAAACCGACGGTTTTGTCGTGACACATGGTACGGATACC ATGGAAGAAACTGCCTATTTCCTCGACCTGACCGTCAAGTGCAACAAGCCGGTAGT GCTGGTGGGTGCAATGCGTCCGTCTACAGGGATGAGCGCCGATGGCCCGTTCAACCT GTATAACGCAGTGGTGACGGCTGCAGACAAAGCCTCTGCCAACCGTGGCGTGCTGG TGGTGATGAACGACACCGTGATGGATGGTCGCGACGTGACCAAAACCAACACTACC GATGTAGCCACCTTCAAATCCGTTAACTATGGCCCGCTGGGCTACATCCATAACGGC AAGATTGACTACCAGCGTACGCCTGCGCGTAAGCACACCACGTCTACTCCGTTCGAT GTGTCTAAGCTGACCGAACTGCCGAAAGTGGGGATTGTTTACAACTACGCTAACGCC TCGGATCTGCCAGCCAAAGCGCTGGTCGACGCGGGTTATGCGGGTATCGTCAGTGC GGGTGTAGGTAACGGCAACTTGTATAAAACGGTATTCGATACGCTGGCCACTGCCG CGCATAAAGGTACCGTCGTGGTGCGTTCCTCCCGTGTACCAACCGGCTCCACCACGC AGGATGCTGAAGTTGATGATGCGAAATACGGCTTTGTGGCTTCAGGTTCTCTGAACC CGCAAAAAGCGCGTGTTCTGCTGCAGCTTGCGCTGACGCAAACCAAGGATCCTAAG CAGATCCAGGAAATGTTTAATCAGTATTAA) of C. werkmanii were copied to relevant positions of ClonExpress Multi S of software CE Design V1.04, and related settings were carried out: a vector was linearized through single enzyme digestion; the number of insert fragments was two; and BglII was selected as an enzyme digestion site for linearization. Output primer pairs ansB-up-F/ansB-up-R and ansa-down-F/ansB-down-R were designed through the software CE Design V1.04 and Guangzhou Branch of Beijing Tsingke Biotechnology Co., Ltd. was entrusted to carry out primer synthesis. Upstream and downstream homologous arms (lanes 2 and 3 in FIG. 1) of the gene ansB were respectively amplified using the primer pairs ansB-up-F/ansB-up-R and ansB-down-F/ansB-down-R and PrimeSTAR® Max DNA Polymerase (TaKaRa) by taking a genome of wild C. werkmanii GDFMZ BF-8 as a template.

(7) Primer sequences were as follows:

(8) ansB-up-F: aaaagtgccacctgcagatctTTCGATATTTGGTGGGACTAAGTAGC (SEQ ID NO: 6);

(9) ansB-up-R: gccacctgcatcgaGTTATTTCTCCAGTTACTTGAATTTGC (SEQ ID NO: 7);

(10) ansB-down-F: aataacTCGATGCAGGTGGCTGCG (SEQ ID NO: 8); and

(11) ansB-down-R: agtcatatgccgcggagatctCGGTCTGGGGCTACGTAGC (SEQ ID NO: 9)

(12) Its mixed system was as follows:

(13) TABLE-US-00001 Reagent Volume (μl) Prime STAR Max Premix (2×) 25 Upstream primer (10 μM) 1 Downstream primer (10 μM) 1 Genome of Citrobacter werkmanii (100 ng/μl) 1 Sterile water 22 Total volume 50

(14) A PCR process was as follows:

(15) TABLE-US-00002 Step Temperature Time (s) Cycle 1 98° C. 10 34 cycles were designed in 2 55° C. 15 total from step 3 72° C. 30 1 to step 3

(16) Products acquired by amplification through the above method were electrophoresed on a 1.0% agarose gel to confirm the correctness of the fragments and recovered corresponding upstream and downstream homologous fragments of ansB by gel cutting.

(17) At the same time, a plasmid pYG4 was extracted with a plasmid extraction kit (Biological Engineering and Biotechnology) and enzyme digestion was carried out using the following enzyme digestion system:

(18) TABLE-US-00003 Reagent Volume (μl) 10 × QuickCut Buffer 5 Plasmid pYG4 (225 ng/ul) 5 BglII (1000 units/ml) 2 Sterile water 38 Total volume 50

(19) BglII used in the above enzyme digestion system was purchased from Takara Biotechnology (Beijing) Co., Ltd. and the uniformly mixed system above was put into an incubator, where it was cultured at 37° C. for 15 minutes, and digested vector fragments were recovered using a gel recovery kit (Omega).

(20) The plasmid pYG4 vector fragments digested and recovered by gel cutting and upstream and downstream homologous arm fragments of the gene ansB were ligated according to instructions of a one-step seamless ligation kit In-Fusion® HD Cloning Kit (TaKaRa):

(21) TABLE-US-00004 Reagent Volume (μl) 5 × in-fusion HD enzyme premix 2 Plasmid pYG4 digested and recovered 4 by gel cutting (42 ng/ul) Upstream homologous fragment 1 ansB-up of ansB (60 ng/ul) Downstream homologous fragment 1 ansB-down of ansB (53 ng/ul) Sterile water 2 Total volume 10

(22) After uniform mixing, the above system was placed in a water bath at 50° C. for 15 minutes, and then placed on ice to terminate the reaction, and 10 μl of entire ligation reaction solution was drawn and transformed with E. coli S17-1 by heat shock (heat shock in a water bath at 42° C. for 90 s). The resulting product was subjected to recovery culture on a shaker for 1 h, spread on a Kana plate and placed in an incubator where it was cultured at 37° C. overnight. After a single colony grew, the single colony was picked and successfully transformed transformants were identified with primers of ansB-QJ-F and ansB-QJ-R (if the length of the amplified fragment was 568 bp, and its sequence was as shown in SEQ ID NO: 5). It was proved that the knockout vector pYG4-ansB was constructed correctly and may be used in subsequent experiments.

(23) ansB-QJ-F: CGCTGGAAAACGATCGTAAAAC (SEQ ID NO: 10); and

(24) ansB-QJ-R: CAAGCCGTTCGAGTTCTTTATG (SEQ ID NO: 11).

(25) II. Conjugational Transfer and ansB Knockout Identification

(26) The E. coli S17-1 carrying the knockout vector pYG4-ansB and the wild type C. werkmanii GDFMZ BF-8 were subjected to conjugational transfer. Specifically, the above two bacteria were respectively cultured overnight, OD.sub.600 was regulated to be equal to 1.0 approximately, and bacterial solutions were mixed according to a volume ratio of 1:3; the mixed bacterial solution was dripped on an LB plate with a filter membrane of 0.22 μm for still standing for 2 h, and after the plate was transferred to an incubator where it was statically cultured at 37° C. for 1 d, the bacteria are eluted with PBS, appropriately diluted and spread on a double-resistant LB plate containing 100 mg/L kanamycin and 20 mg/L rifampicin for culture at 37° C. for 1-2 d. A growing colony was picked and subjected to PCR verification by using the primers ansB-QJ-F and ansB-QJ-R. A recombinant of the gene ansB acquired by one-time exchange should have two bands: a large band of 1615 bp and a small band of 568 bp.

(27) The strains that have been successfully recombined in one exchange were subjected to amplification culture in LB liquid medium, appropriately diluted with an amplification culture bacteria solution and then streaked on an LB plate containing 5% sucrose by mass fraction. After culture for 72 h, a single colony on the plate was picked for PCR verification (lane 4 in FIG. 1) by using primers ansB-QJ-F and ansB-QJ-R to determine ansB knockout strains. The knockout strains should have been subjected to double exchange, and so only a small band may be amplified, that is, a 568-bp band (its sequence was as shown in SEQ ID NO: 5). The colony identified as positive by PCR was streaked on the LB plate containing 100 mg/L kanamycin or the LB plate containing 20 mg/L rifampicin, respectively and rifampicin-resistant and kanamycin-sensitive strains finally acted as gene ansB knockout strain of ΔansB for subsequent experiments.

(28) The gene ansB knockout strain was designated as C. werkmanii ΔansB, and deposited in Guangdong Microbial Culture Collection Center (GDMCC) on the 5th Floor, Building NO, 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, 510070 under the accession No. GDMCC 61849 on Aug. 2, 2021.

(29) III. Determination of Biofilm Formation Capacity of ansB Knockout Mutant

(30) The biofilm formation capacity of ΔansB was determined by using three different media, i.e., a common LB medium, a nutrient broth (NB) medium and a tryptone soy broth (TSB) medium, at three temperatures (25° C., 30° C. and 37° C.). The main experimental steps were as follows: ΔansB and the wild type of C. werkmanii GDFMZ BF-8 were respectively cultured overnight in LB, NB and TSB, and on the second day, the concentration of each bacterial solution was adjusted to OD.sub.600=1.0 with fresh LB, NB and TSB respectively for later use; 180 μl of fresh sterile LB medium, 180 μl of fresh sterile NB medium and 180 μl of fresh sterile TSB medium were respectively added to 96-well plates (Corning), and then 20 μl of the bacterial solution, of which the bacterial concentration was adjusted in advance was added; after the above 96-well plates, to which samples were added, were respectively placed into incubators with the temperatures of 25° C., 30° C. and 37° C. for static culture or shake culture (120 rpm) for 2 days, firstly planktonic bacteria were discarded and the 96-well plates were washed, dyeing was carried out with 0.1% crystal violet; after excess dye was eluted with sterile water, crystal violet remaining on inner well walls of the 96-well plates were eluted using 95% alcohol, light absorption values of the samples at 590 nm were determined with an enzyme-labeled instrument and were used to represent the biofilm formation. 8 repeats were set in each treatment and the treatment was repeated at least 3 times at different time.

(31) The biofilm formation capacities of the wild type strain of C. werkmanii GDFMZ BF-8 and the ansB knockout mutant of ΔansB under different conditions were shown in FIGS. 2A-2F. Compared with the wild type strain of C. werkmanii GDFMZ BF-8, the increased multiples of the biofilm formation capacity of ΔansB under different conditions were shown in the following table.

(32) TABLE-US-00005 Medium Temperature Culture mode LB NB TSB 25° Static culture 2.15 1.67 1.49 Shake static 1.36 1.11 1.55 30° Static culture 2.68 1.37 1.73 Shake static 1.64 0.93 0.92 37° Static culture 1.15 1.23 1.03 Shake static 1.26 0.80 0.79

(33) It can be seen from FIGS. 2A-2F and the above table that the wild strain of C. werkmanii GDFMZ BF-8 may form more biofilms at a relatively high temperature (37° C.) under a static condition (FIG. 2C), but forms less biofilms at a relatively low temperature (25° C. and 30° C.) (FIGS. 2A and 2B). However, the biofilm formation capacity of ΔansB was improved in the LB medium under both static and shake cultures at 25° C., 30° C. and 37° C., was optimal at 30° C. under static culture, and improved by 2.68 times in total; was optimal at 25° C. in the NB medium under static culture (improved by 1.67 times); and was optimal at 30° C. in the TSB medium under static culture (improved by 1.73 times). In summary, ΔansB can form more biofilms than the wild strain of C. werkmanii GDFMZ BF-8 under the conditions of a polypropylene attachment material, the LB medium, 30° C., and static culture.

(34) The above results showed that the biofilm formation of this strain can be improved by knocking out the gene ansB of the Citrobacter werkmanii (the required optimized conditions: polypropylene attachment material, LB medium, 30° C. and static culture), and the strain has practical application potential and prospects under specific conditions.