CN104894154B - The Ts2 of responsive to temperature type carrier pBBR1MCS 2 and its application - Google Patents
The Ts2 of responsive to temperature type carrier pBBR1MCS 2 and its application Download PDFInfo
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Abstract
The invention discloses the Ts2 of responsive to temperature type carrier pBBR1MCS 2 and its application.The Ts2 of responsive to temperature type carrier pBBR1MCS 2 provided by the present invention, are that the DNA fragmentation shown in the 1557th 2526 of SEQ ID No.1 is replaced with into destination gene expression box, and keep SEQ ID No.1 other invariant nucleotides, obtained recombinant vector；The protein of destination gene expression box coding is that the protein shown in SEQ ID No.2 is carried out into A2) transformation, keep the SEQ ID No.2 constant obtained protein of other amino acid residues：The A2) it is that SEQ ID No.2 the 130th His is sported into Gln.
Description
Technical field
The present invention relates to responsive to temperature type carrier pBBR1MCS-2-Ts2 and its application in biological technical field.
Background technology
One section of useful target DNA fragment by DNA recombinant techniques, it is sent into recipient cell and goes to be replicated and expressed
Instrument carrier (Vector).Plasmid is the carrier commonly used in recombinant DNA technology, and plasmid is present in many bacteriums and yeast
It is the ring-shaped DNA molecule for the very little for being capable of autonomous replication outside cell chromosome in the biology such as bacterium.One preferable carrier substantially should
There are some following characteristics：(1) molecular weight is small, multicopy, relaxation control type；(2) with a variety of conventional restriction enzymes
Single point of contact；(3) larger exogenous dna fragment can be inserted；(4) there is genetic marker, be easy to identify and screen；(5) it is thin to host
Born of the same parents are harmless.
Wide host range plasmid pBBR1MCS-2 is the very wide carrier of application, and the plasmid is transformed by pBBR1 plasmids.
PBBR1 plasmid origins are in Gram-negative bacteria Bordetella bronchiseptica S87 (Antoine R and Locht
C.Isolation and molecular characterization of a novel broad-host-range
plasmid from Bordetella bronchiseptica with sequence similarities to plasmids
from Gram-positive organisms.Mol Microbiol.1992,6(13):1785-1799).PBBR1MCS-2 contains
There is kanamycins (kan) resistance screening mark, its size only has 5145bp, and these characteristics cause the plasmid to be applied to
DNA clone, protein expression, it is widely used in the molecule manipulation of Gram-negative bacteria.
Temperature-sensitive plasmid carrier can easily control the presence of plasmid in host.When temperature than it is relatively low when, matter
Grain may have in host, and after temperature improves, temperature-sensitive plasmid carrier will be lost or is incorporated on chromosome.
The content of the invention
The technical problems to be solved by the invention are how to build responsive to temperature type carrier.
In order to solve the above technical problems, present invention firstly provides the method for structure recombinant vector.
The method of structure recombinant vector provided by the present invention, is by SEQ ID to build the method 1 of recombinant vector
DNA fragmentation shown in No.1 1557-2526 positions replaces with destination gene expression box, and keeps other of SEQ ID No.1
Invariant nucleotide, obtained recombinant DNA are the recombinant vector, and the recombinant vector is named as into pBBR1MCS-2-Ts2；
The protein of destination gene expression box coding is that the protein shown in SEQ ID No.2 is carried out into A2) change
The SEQ ID No.2 constant obtained protein of other amino acid residues is made, keeps, i.e., described destination gene expression box coding
Protein be the protein shown in SEQ ID No.6；The A2) it is to sport SEQ ID No.2 the 130th His
Gln。
In the above method 1, the target gene in the destination gene expression box is by SEQ ID No.1 1794-
DNA molecular shown in 2456 nucleotides carries out B2) transformation, keep SEQ ID No.1 1794-2456 positions nucleotides institute
The constant obtained DNA molecular of other nucleotides in the DNA molecular shown；The B2) it is by SEQ ID No.1's the 2183rd
C sports G.
Wherein, SEQ ID No.1 are made up of 5145 nucleotides, and SEQ ID No.1 1794-2456 positions nucleotides is
Rep encoding gene, the rep albumen shown in coding SEQ ID No.2.
In the above method 1, the sequence of the destination gene expression box can be SEQ ID No.5.
Wherein, the protein shown in SEQ ID No.5 238-900 positions coding SEQ ID No.6.
In the above method 1, the promoter for starting the destination gene expression in the destination gene expression box is by SEQ
DNA molecular shown in ID No.1 1557-1793 positions carries out following C2), C3) and C4) at least one of these three change
Make, keep other nucleotides in DNA molecular shown in SEQ ID No.1 1557-1793 positions nucleotides are constant to obtain
DNA molecular：
C2) following C21) and/or C22)：
C21 SEQ ID No.1 the 1732nd A) is sported into G；
C22 SEQ ID No.1 the 1780th G) is sported into T；
C3) following C31) and/or C32)：
C31 SEQ ID No.1 the 1747th C) is sported into T；
C32 SEQ ID No.1 the 1776th T) is sported into A；
C4 SEQ ID No.1 the 1751st T) is sported into A；
The terminator that the destination gene expression is terminated in the destination gene expression box is the of SEQ ID No.1
DNA molecular shown in 2457-2526 positions.
In order to solve the above technical problems, present invention also offers the method for structure recombinant vector.
The method of structure recombinant vector provided by the present invention, is by SEQ ID to build the method 2 of recombinant vector
DNA fragmentation shown in No.1 1557-2526 positions replaces with destination gene expression box, and keeps other of SEQ ID No.1
Invariant nucleotide, obtained recombinant DNA are the recombinant vector；
The protein of the destination gene expression box coding is that the protein shown in SEQ ID No.2 is carried out into the A2)
And following A 3) and A4) transformation of at least one of both, holding SEQ ID No.2 other amino acid residues do not become
To the obtained protein of protein：
A3 SEQ ID No.2 the 86th Arg) is sported into Cys；
A4) following A41) and/or A42)：
A41 SEQ ID No.2 the 25th Lys) is sported into Glu；
A42 SEQ ID No.2 the 146th Ile) is sported into Val.
In the above method 2, the target gene in the destination gene expression box is by SEQ ID No.1 1794-
DNA molecular shown in 2456 nucleotides carries out the B2) and following B3) and the B4) transformation, holding of at least one of both
Constant obtained DNA points of other nucleotides in DNA molecular shown in SEQ ID No.1 1794-2456 positions nucleotides
Son：
B3 SEQ ID No.1 the 2049th C) is sported into T；
B4) following B41) and/or B42)：
B41 SEQ ID No.1 the 1866th A) is sported into G；
B42 SEQ ID No.1 the 2229th A) is sported into G.
In the above method 2, the promoter for starting the destination gene expression in the destination gene expression box is by SEQ
DNA molecular shown in ID No.1 1557-1793 positions carries out the C2), the C3) and the C4) at least one in these three
Other nucleotides in DNA molecular shown in the transformation of kind, holding SEQ ID No.1 1557-1793 positions nucleotides are not
Become obtained DNA molecular；
The terminator that the gene expression is terminated in the expression casette is SEQ ID No.1 2457-2526 positions
Shown DNA molecular.
In order to solve the above technical problems, present invention also offers the restructuring built by methods described 1 or methods described 2 load
Body.
In order to solve the above technical problems, present invention also offers the recombinant microorganism containing the recombinant vector or restructuring are thin
Born of the same parents system.
The recombinant microorganism concretely bacterium, yeast, algae and fungi.Wherein, the bacterium can be Escherichia coli or
Hydrogenlike silicon ion.The Escherichia coli concretely Escherichia coli Escherichia coli T1, Escherichia coli
Escherichia coli DH5 α, Escherichia coli Escherichia coli BW25113 or Escherichia coli Escherichia
coli S17-1.The hydrogenlike silicon ion concretely hydrogenlike silicon ion Rhodobacter sphaeroides 2.4.1.
The recombinant cell lines do not include propagating materials.
In order to solve the above technical problems, present invention also offers following any products：
P1, the protein；
P2, the target gene；
P3, DNA molecular, for the DNA molecular shown in SEQ ID No.1 1557-1793 positions is carried out into the C2) or under
State Z234) transformation, keep other nucleotides in DNA molecular shown in SEQ ID No.1 1557-1793 positions nucleotides
Constant obtained DNA molecular；
At least one of Z234) it is the C2) and the C3) and the C4) both.
P3, DNA molecular, for the DNA molecular shown in SEQ ID No.1 1557-1793 positions is carried out into the C2) and institute
State C3) and the C4) both at least one of transformation, keep shown in SEQ ID No.1 1557-1793 positions nucleotides
DNA molecular in the constant obtained DNA molecular of other nucleotides.
In order to solve the above technical problems, present invention also offers following any applications：
The application of L1, the target gene or the DNA molecular in preparation temperature sensitive carrier；
The application of L2, the DNA molecular in as promoter；
The application of L3, the recombinant vector in as temperature sensitive vector.
Application described in above-mentioned L3 can be that the recombinant vector carries in Escherichia coli or hydrogenlike silicon ion as temperature sensitivity
Application in body.The Escherichia coli concretely Escherichia coli Escherichia coli T1, Escherichia coli
Escherichia coli DH5 α, Escherichia coli Escherichia coli BW25113 or Escherichia coli Escherichia
coli S17-1.The hydrogenlike silicon ion concretely hydrogenlike silicon ion Rhodobacter sphaeroides 2.4.1.
In the application, the temperature sensitive vector is that the carrier may be present in host at a certain temperature, and is changed
After temperature, the carrier can not be present in the host.Concretely the carrier may be present in host at 30 DEG C, when
When temperature is 37 DEG C -42 DEG C, the carrier can not be present in the host.
It is demonstrated experimentally that the recombinant vector pBBR1MCS-2-Ts2 of the present invention is temperature sensitive vector：It is respectively in Host Strains
Escherichia coli Escherichia coli T1, Escherichia coli Escherichia coli DH5 α, Escherichia coli Escherichia
During coli BW25113 and Escherichia coli Escherichia coli S17-1, pBBR1MCS-2 and pBBR1MCS-2-Ts2 are 30
DEG C LB fluid nutrient mediums and Loss Rate in 30 DEG C of LB+kan fluid nutrient mediums and pBBR1MCS-2 are in 42 DEG C of LB Liquid Cultures
Loss Rate in base is below 10%, and Loss Rates of the pBBR1MCS-2-Ts2 in 42 DEG C of LB fluid nutrient mediums reaches
99.99%；When Host Strains are hydrogenlike silicon ion Rhodobacter sphaeroides 2.4.1, pBBR1MCS-2 and
Loss Rates and pBBR1MCS- of the pBBR1MCS-2-Ts2 in 30 DEG C of LB fluid nutrient mediums and 30 DEG C of LB+kan fluid nutrient mediums
2 Loss Rate in 37 DEG C of LB fluid nutrient mediums is below 13%, and pBBR1MCS-2-Ts2 is in 37 DEG C of LB fluid nutrient mediums
In Loss Rate reach 81.36%.
It is demonstrated experimentally that the recombinant vector pBBR1MCS-2-Ts2 of the present invention can be applied to for temperature sensitive vector：(1) carrier
Eliminate, i.e., remove carrier by improving temperature；(2) single crossover homologous recombinates, i.e., by improving temperature, allows carrier DNA to be incorporated into
The homology region of chromosome；(3) double crossing over homologous recombination, i.e., by improving temperature, carrier DNA is allowed to replace chromosome the preceding paragraph
DNA。
Brief description of the drawings
Fig. 1 is pBBR1MCS-2 collection of illustrative plates.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments, it is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Wide host cell pBBR1MCS-2 in following embodiments is Biovector China plasmid vector strain cell strain base
Because of collection product.
Escherichia coli Escherichia coli T1 in following embodiments are Beijing Quanshijin Biotechnology Co., Ltd
Product, catalog number (Cat.No.) CD501-01.
Escherichia coli Escherichia coli DH5 α in following embodiments are precious bioengineering (Dalian) limited public affairs
Department, catalog number (Cat.No.) 9057.
Escherichia coli Escherichia coli BW25113 in following embodiments are Biovector China plasmid vector
Strain cell pnca gene collection product.
Escherichia coli Escherichia coli S17-1 in following embodiments are Biovector China plasmid vector bacterium
Strain cell pnca gene collection product.
Hydrogenlike silicon ion Rhodobacter sphaeroides 2.4.1 in following embodiments receive for American Type Culture
Collection center (ATCC) product, bacterial strain number are 17023.
Sistrom culture mediums in following embodiments are in document (Sistrom WR.The kinetics of the
synthesis of photopigments in Rhodopseudomonas
sphaeroides.J.Gen.Microbiol.1962.28:Reported in 607-616).
LB fluid nutrient mediums in following embodiments are to form aseptic culture medium by solute and solvent, and solvent is water, solute
And its concentration is respectively tryptone 1g/100mL, yeast extract 0.5g/100mL, NaCl 0.5g/100mL；
LB+kan fluid nutrient mediums are that the aseptic culture medium that kanamycins obtains, LB+kan are added into LB fluid nutrient mediums
The content of kanamycins is 50mg/L in fluid nutrient medium；
LB flat boards are that the sterile solid culture medium that agar obtains is added into LB fluid nutrient mediums；
LB+kan flat boards are that the sterile solid culture medium that agar and kanamycins obtain, LB are added into LB fluid nutrient mediums
The content of kanamycins is 50mg/L in+kan flat boards.
The preparation of embodiment 1, temperature sensitive vector
1st, the screening of temperature sensitive vector
To the DNA fragmentation shown in the 1557-2526 positions of SEQ ID No.1 in wide host cell pBBR1MCS-2 (Fig. 1)
Random mutation is carried out, obtains the DNA molecular after the mutation of a plurality of different random mutation；By SEQ ID No.1 in pBBR1MCS-2
1557-2526 positions shown in DNA fragmentation replace with the DNA molecular after above-mentioned each mutation respectively, keep pBBR1MCS-2's
Other sequences are constant, obtain multiple recombinant vectors containing mutated DNA fragment；These restructuring for containing mutated DNA fragment are carried
Body is directed respectively into Escherichia coli Escherichia coli T1, obtains 1068 restructuring large intestine bars containing mutated DNA fragment
Bacterium.PBBR1MCS-2 is imported in Escherichia coli Escherichia coli T1, obtains recombination bacillus coli T1 (pBBR1MCS-
2)。
Wherein, the rep albumen shown in SEQ ID No.1 1794-2456 positions nucleotide coding SEQ ID No.2.
The above-mentioned recombination bacillus coli containing mutated DNA fragment is screened by the following method, and by T1
(pBBR1MCS-2) as control：The same clone of each recombination bacillus coli in this 1068 recombination bacillus colis is distinguished
Carry out processing 1. and 2.：1. recombination bacillus coli is seeded on LB flat boards, 18h is cultivated at 42 DEG C, then by LB flat boards
The recombination bacillus coli grown at upper 42 DEG C is seeded on LB+kan flat boards, and 18h is cultivated at 42 DEG C；2. by recombination bacillus coli
It is seeded on LB+kan flat boards, 18h is cultivated at 30 DEG C.The flat board of LB+kan at 42 DEG C and 30 DEG C of contrast, discovery have 4 plant weights
Group Escherichia coli grow normally on the LB+Kan flat boards at 30 DEG C, but can not grow (table on the LB+Kan flat boards at 42 DEG C
1), and on the LB+Kan flat boards at LB+Kan flat boards and 42 DEG C of the T1 (pBBR1MCS-2) at 30 DEG C can normal growth, table
Bright, this 4 plants of recombination bacillus colis are temperature sensitive.The weight that will be mutated in this 4 plants of recombination bacillus colis from pBBR1MCS-2
Group carrier is respectively designated as pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4,
Corresponding recombination bacillus coli is respectively designated as T1 (pBBR1MCS-2-Ts 1), T1 (pBBR1MCS-2-Ts2), T1
And T1 (pBBR1MCS-2-Ts4) (pBBR1MCS-2-Ts3).
The screening of table 1, recombination bacillus coli
| Transformant | 30 DEG C of LB+Kan flat boards | 42 DEG C of LB+Kan flat boards | Temperature is sensitive |
| T1(pBBR1MCS-2-Ts1) | + | - | + |
| T1(pBBR1MCS-2-Ts2) | + | - | + |
| T1(pBBR1MCS-2-Ts3) | + | - | + |
| T1(pBBR1MCS-2-Ts4) | + | - | + |
| T1(pBBR1MCS-2) | + | + | - |
Extract T1 (pBBR1MCS-2-Ts1), T1 (pBBR1MCS-2-Ts2), T1 (pBBR1MCS-2-Ts3) and T1
(pBBR1MCS-2-Ts4) pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2- in
Ts4 plasmids, respectively to pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 couple
DNA fragmentation that should be shown in the 1557-2526 positions nucleotides in SEQ ID No.1 is sequenced, and is found：
PBBR1MCS-2-Ts1 is that pBBR1MCS-2 is carried out into following B11), B12), B13) and transformation B14) and holding
The constant obtained recombinant vector of pBBR1MCS-2 other sequences, pBBR1MCS-2-Ts1 1557-2526 positions nucleotides sequence
It is classified as SEQ ID No.3：
B11 SEQ ID No.1 the 1827th G) is sported into A；
B12 SEQ ID No.1 the 2034th G) is sported into C；
B13 SEQ ID No.1 the 2212nd A) is sported into T；
B14 SEQ ID No.1 the 2249th C) is sported into A.
PBBR1MCS-2-Ts2 is that pBBR1MCS-2 is carried out into following B2), C21) and transformation C22) and holding
The constant obtained recombinant vector of pBBR1MCS-2 other sequences, pBBR1MCS-2-Ts2 1557-2526 positions nucleotides sequence
It is classified as SEQ ID No.5：
B2 SEQ ID No.1 the 2183rd C) is sported into G；
C21 SEQ ID No.1 the 1732nd A) is sported into G；
C22 SEQ ID No.1 the 1780th G) is sported into T；
PBBR1MCS-2-Ts3 is that pBBR1MCS-2 is carried out into following B3), C31) and transformation C32) and holding
The constant obtained recombinant vector of pBBR1MCS-2 other sequences, pBBR1MCS-2-Ts3 1557-2526 positions nucleotides sequence
It is classified as SEQ ID No.7：
B3 it is) that SEQ ID No.1 the 2049th C is sported into T；
C31 SEQ ID No.1 the 1747th C) is sported into T；
C32 SEQ ID No.1 the 1776th T) is sported into A；
PBBR1MCS-2-Ts4 is that pBBR1MCS-2 is carried out into following B41), B42) and transformation C4) and holding
The constant obtained recombinant vector of pBBR1MCS-2 other sequences, pBBR1MCS-2-Ts4 1557-2526 positions nucleotides sequence
It is classified as SEQ ID No.9：
B41 SEQ ID No.1 the 1866th A) is sported into G；
B42 SEQ ID No.1 the 2229th A) is sported into G；
C4 SEQ ID No.1 the 1751st T) is sported into A.
Show, compared with pBBR1MCS-2, pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and
There occurs the mutation of partial nucleotide by pBBR1MCS-2-Ts4.
Except T1 (pBBR1MCS-2-Ts1), T1 (pBBR1MCS-2-Ts2), T1 in 1068 recombination bacillus colis
And outer recombination bacillus coli (the LB+Kan flat boards and 42 at 30 DEG C of T1 (pBBR1MCS-2-Ts4) (pBBR1MCS-2-Ts3)
On LB+Kan flat boards at DEG C can normal growth, i.e., temperature-resistant recombination bacillus coli) in randomly select 10 plants, it is right
It is sequenced, is as a result found, this 10 plants temperature-resistant from the recombinant vector of pBBR1MCS-2 mutation in this 10 plants
From the recombinant vector of pBBR1MCS-2 mutation, also there occurs the mutation of partial nucleotide in recombination bacillus coli.
Compared with pBBR1MCS-2, the protein (amino of pBBR1MCS-2-Ts1 1794-2456 positions nucleotide coding
Acid sequence is SEQ ID No.4) be that the protein shown in SEQ ID No.2 is subjected to following A11), A12) and transformation A13)
Obtained protein：
A11 SEQ ID No.2 the 12nd Ala) is sported into Thr；
A12 SEQ ID No.2 the 81st Asp) is sported into His；
A13 SEQ ID No.2 the 140th His) is sported into Leu.
Compared with pBBR1MCS-2, the protein (amino of pBBR1MCS-2-Ts2 1794-2456 positions nucleotide coding
Acid sequence is SEQ ID No.6) be that the protein shown in SEQ ID No.2 is subjected to following A2) the obtained protein of transformation：
A2 it is) that SEQ ID No.2 the 130th His is sported into Gln.
Compared with pBBR1MCS-2, the protein (amino of pBBR1MCS-2-Ts3 1794-2456 positions nucleotide coding
Acid sequence is SEQ ID No.8) be that the protein shown in SEQ ID No.2 is subjected to following A3) the obtained protein of transformation：
A3 it is) that SEQ ID No.2 the 86th Arg is sported into Cys.
Compared with pBBR1MCS-2, the protein (amino of pBBR1MCS-2-Ts4 1794-2456 positions nucleotide coding
Acid sequence is SEQ ID No.10) be that the protein shown in SEQ ID No.2 is subjected to following A41) and transformation A42) obtain
Protein：
A41 SEQ ID No.2 the 25th Lys) is sported into Glu；
A42 SEQ ID No.2 the 146th Ile) is sported into Val.
2nd, pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 temperature are quick
The checking of perception
The T1 (pBBR1MCS-2-Ts1) of step 1 is seeded in LB+kan fluid nutrient mediums, cultivated 18 hours in 30 DEG C,
Obtained bacterium solution is subjected to following handle：
1. bacterium solution is pressed 1：1000 ratio is seeded in 3mL LB fluid nutrient mediums, and then culture 18 is small at 30 DEG C
When, obtain LB-30 DEG C of bacterium solution；LB-30 DEG C of dilution bacterium solution is obtained after LB-30 DEG C of bacterium solution is diluted, by LB-30 DEG C of dilution bacterium solution point
It is not applied on LB flat boards and LB+kan flat boards, each μ L LB-30 DEG C of flat board 100 dilute bacterium solution, then cultivate 18 at 30 DEG C
Hour, LB-LB-30 DEG C of flat board and LB-LB+kan-30 DEG C of flat board are respectively obtained, counts LB-LB-30 DEG C of flat board and LB-LB+
Clump count on kan-30 DEG C of flat board, recombinant vector pBBR1MCS-2-Ts1 losing in 30 DEG C of LB fluid nutrient mediums is calculated
Mistake rate (table 2), Loss Rate=(bacterium colony on-LB-LB+kan-30 DEG C of flat boards of clump count on LB-LB-30 DEG C of flat board of carrier
Number) clump count on ÷ LB-LB-30 DEG C flat boards；
2. bacterium solution is pressed 1：1000 ratio is seeded in 3mL LB+kan fluid nutrient mediums, then cultivates 18 at 30 DEG C
Hour, obtain LB+kan-30 DEG C of bacterium solution；LB+kan-30 DEG C of dilution bacterium solution is obtained after LB+kan-30 DEG C of bacterium solution is diluted, by LB+
Kan-30 DEG C of dilution bacterium solution is respectively coated to LB flat boards and LB+kan flat boards, each μ L LB+kan-30 DEG C of flat board 100 dilutions
Bacterium solution, then cultivated 18 hours at 30 DEG C, respectively obtain LB+kan-LB-30 DEG C of flat board and LB+kan-LB+kan-30 DEG C flat
Plate, the clump count on LB+kan-LB-30 DEG C of flat board and LB+kan-LB+kan-30 DEG C of flat board is counted, recombinant vector is calculated
Loss Rates (table 2) of the pBBR1MCS-2-Ts1 in 30 DEG C of LB+kan fluid nutrient mediums, Loss Rate=(LB+kan-LB- of carrier
The clump count on-LB+kan-LB+kan-30 DEG C of flat boards of clump count on 30 DEG C of flat boards) on ÷ LB+kan-LB-30 DEG C flat boards
Clump count；
3. bacterium solution is pressed 1：1000 ratio is seeded in 3mL LB fluid nutrient mediums, and then culture 18 is small at 42 DEG C
When, obtain LB-42 DEG C of bacterium solution；LB-42 DEG C of dilution bacterium solution is obtained after LB-42 DEG C of bacterium solution is diluted, by LB-42 DEG C of dilution bacterium solution point
It is not applied on LB flat boards and LB+kan flat boards, each μ L LB-42 DEG C of flat board 100 dilute bacterium solution, then cultivate 18 at 42 DEG C
Hour, LB-LB-42 DEG C of flat board and LB-LB+kan-42 DEG C of flat board are respectively obtained, counts LB-LB-42 DEG C of flat board and LB-LB+
Clump count on kan-42 DEG C of flat board, recombinant vector pBBR1MCS-2-Ts1 losing in 42 DEG C of LB fluid nutrient mediums is calculated
Mistake rate (table 2), Loss Rate=(bacterium colony on-LB-LB+kan-42 DEG C of flat boards of clump count on LB-LB-42 DEG C of flat board of carrier
Number) clump count on ÷ LB-LB-42 DEG C flat boards.
According to the method described above, T1 (pBBR1MCS-2-Ts1) is replaced with into T1 (pBBR1MCS-2-Ts2), T1
(pBBR1MCS-2-Ts3), T1 (pBBR1MCS-2-Ts4) and T1 (pBBR1MCS-2), other steps are constant, obtain restructuring and carry
Body pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3, pBBR1MCS-2-Ts4 and pBBR1MCS-2 train in 30 DEG C of LB liquid respectively
Support the Loss Rate (table 2) in base, 30 DEG C of LB+kan fluid nutrient mediums and 42 DEG C of LB fluid nutrient mediums.
The Loss Rate of table 2, recombinant vector at different temperatures
Above-mentioned pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are 30
Between 8.70%-9.62%, the Loss Rate in 30 DEG C of LB+kan fluid nutrient mediums exists Loss Rate in DEG C LB fluid nutrient mediums
Between 4.44%-7.84%, and the Loss Rate in 42 DEG C of LB fluid nutrient mediums is up to 99.99%, and pBBR1MCS-2 this three
Loss Rate in kind of fluid nutrient medium between 4.76%-9.80%, illustrate pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2,
PBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 is responsive to temperature type carrier.
Embodiment 2, pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4
Temperature sensitivity in Escherichia coli Escherichia coli DH5 α
Respectively by pBBR1MCS-2 and pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2 of the step 1 of embodiment 1,
PBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 is directed respectively into Escherichia coli Escherichia coli DH5 α, respectively
To recombination bacillus coli DH5 α (pBBR1MCS-2), DH5 α (pBBR1MCS-2-Ts1), DH5 α (pBBR1MCS-2-Ts2), DH5 α
And DH5 α (pBBR1MCS-2-Ts4) (pBBR1MCS-2-Ts3).
According to the method for the step 2 of embodiment 1, T1 (pBBR1MCS-2-Ts1) is replaced with into above-mentioned DH5 α respectively
(pBBR1MCS-2), DH5 α (pBBR1MCS-2-Ts1), DH5 α (pBBR1MCS-2-Ts2), DH5 α (pBBR1MCS-2-Ts3) and
DH5 α (pBBR1MCS-2-Ts4), other steps are constant, and it is Escherichia coli Escherichia coli to respectively obtain Host Strains
During DH5 α recombinant vector pBBR1MCS-2, pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and
PBBR1MCS-2-Ts4 is respectively in 30 DEG C of LB fluid nutrient mediums, 30 DEG C of LB+kan fluid nutrient mediums and 42 DEG C of LB fluid nutrient mediums
Loss Rate (table 3).
The Loss Rate of table 3, responsive to temperature type carrier in Escherichia coli Escherichia coli DH5 α
When Host Strains are Escherichia coli Escherichia coli DH5 α, pBBR1MCS-2, pBBR1MCS-2-Ts1,
PBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are in 30 DEG C of LB fluid nutrient mediums and 30 DEG C of LB+kan liquid
The Loss Rate of Loss Rate and pBBR1MCS-2 in 42 DEG C of LB fluid nutrient mediums in body culture medium below 10%,
PBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are in 42 DEG C of LB Liquid Cultures
Loss Rate in base reaches 99.99%, illustrate pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and
PBBR1MCS-2-Ts4 can show its temperature sensitivity in Escherichia coli Escherichia coli DH5 α.
Embodiment 3, pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4
Temperature sensitivity in Escherichia coli Escherichia coli BW25113
Respectively by pBBR1MCS-2 and pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2 of the step 1 of embodiment 1,
PBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 is directed respectively into Escherichia coli Escherichia coli BW25113, respectively
Obtain recombination bacillus coli BW25113 (pBBR1MCS-2), BW25113 (pBBR1MCS-2-Ts1), BW25113 (pBBR1MCS-
2-Ts2), BW25113 (pBBR1MCS-2-Ts3) and BW25113 (pBBR1MCS-2-Ts4).
According to the method for the step 2 of embodiment 1, T1 (pBBR1MCS-2-Ts1) is replaced with into above-mentioned BW25113 respectively
(pBBR1MCS-2)、BW25113(pBBR1MCS-2-Ts1)、BW25113(pBBR1MCS-2-Ts2)、BW25113
(pBBR1MCS-2-Ts3) and BW25113 (pBBR1MCS-2-Ts4), other steps are constant, and it is large intestine to respectively obtain Host Strains
Recombinant vector pBBR1MCS-2, pBBR1MCS-2-Ts1, pBBR1MCS-2- during bacillus Escherichia coli BW25113
Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are respectively in 30 DEG C of LB fluid nutrient mediums, 30 DEG C of LB+kan fluid nutrient mediums
With the Loss Rate (table 4) in 42 DEG C of LB fluid nutrient mediums.
The Loss Rate of table 4, responsive to temperature type carrier in Escherichia coli Escherichia coli BW25113
When Host Strains are Escherichia coli Escherichia coli BW25113, pBBR1MCS-2, pBBR1MCS-2-
Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are in 30 DEG C of LB fluid nutrient mediums and 30 DEG C of LB+
The Loss Rate of Loss Rate and pBBR1MCS-2 in 42 DEG C of LB fluid nutrient mediums in kan fluid nutrient mediums 10% with
Under, pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 train in 42 DEG C of LB liquid
The Loss Rate supported in base reaches 99.99%, illustrates pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3
With pBBR1MCS-2-Ts4 its temperature sensitivity can be shown in Escherichia coli Escherichia coli BW25113.
Embodiment 4, pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4
Temperature sensitivity in Escherichia coli Escherichia coli S17-1
Respectively by pBBR1MCS-2 and pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2 of the step 1 of embodiment 1,
PBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 is directed respectively into Escherichia coli Escherichia coli S17-1, respectively
To recombination bacillus coli S17-1 (pBBR1MCS-2), S17-1 (pBBR1MCS-2-Ts1), S17-1 (pBBR1MCS-2-Ts2),
S17-1 (pBBR1MCS-2-Ts3) and S17-1 (pBBR1MCS-2-Ts4).
According to the method for the step 2 of embodiment 1, T1 (pBBR1MCS-2-Ts1) is replaced with into above-mentioned S17-1 respectively
(pBBR1MCS-2)、S17-1(pBBR1MCS-2-Ts1)、S17-1(pBBR1MCS-2-Ts2)、S17-1(pBBR1MCS-2-
Ts3) and S17-1 (pBBR1MCS-2-Ts4), other steps are constant, and it is Escherichia coli to respectively obtain Host Strains
Recombinant vector pBBR1MCS-2, pBBR1MCS-2-Ts1 during Escherichia coli S17-1, pBBR1MCS-2-Ts2,
PBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 is respectively in 30 DEG C of LB fluid nutrient mediums, 30 DEG C of LB+kan fluid nutrient mediums and 42
Loss Rate (table 5) in DEG C LB fluid nutrient mediums.
The Loss Rate of table 5, responsive to temperature type carrier in Escherichia coli Escherichia coli S17-1
When Host Strains are Escherichia coli Escherichia coli S17-1, pBBR1MCS-2, pBBR1MCS-2-Ts1,
PBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are in 30 DEG C of LB fluid nutrient mediums and 30 DEG C of LB+kan liquid
The Loss Rate of Loss Rate and pBBR1MCS-2 in 42 DEG C of LB fluid nutrient mediums in body culture medium below 10%,
PBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are in 42 DEG C of LB Liquid Cultures
Loss Rate in base reaches 99.99%, illustrate pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and
PBBR1MCS-2-Ts4 can show its temperature sensitivity in Escherichia coli Escherichia coli S17-1.
Embodiment 5, pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4
Temperature sensitivity in hydrogenlike silicon ion Rhodobacter sphaeroides 2.4.1
1st, the preparation of hydrogenlike silicon ion is recombinated
(1) the recombination bacillus coli S17-1 (pBBR1MCS-2) of embodiment 4 is shaken for 37 DEG C in LB+kan fluid nutrient mediums
Bottle is incubated overnight, and next day is forwarded to fresh LB fluid nutrient mediums with 1/10 switching amount, continues to cultivate about 1-2 hours extremely
OD600 is 1.0 or so, obtains S17-1 (pBBR1MCS-2) nutrient solution；Take 1.5mL S17-1 (pBBR1MCS-2) nutrient solution
3-5 minutes are centrifuged under 7000rpm, supernatant is abandoned, obtains bacterial sediment, are precipitated 2 times with fresh LB culture mediums washing thalline, are used
Bacterial sediment is resuspended in 200 μ L LB fluid nutrient mediums, obtains donor bacterium suspension；
(2) hydrogenlike silicon ion Rhodobacter sphaeroides 2.4.1 are trained in LB fluid nutrient mediums at 30 DEG C
It is 1.7-2.5 or so to be forwarded to fresh LB culture mediums after supporting 24 hours to continue culture about 24 hours to OD600, and it is red to obtain class ball
Inoculum；Centrifugation 3-5 minutes under 1.5mL hydrogenlike silicon ion nutrient solutions 7000rpm are taken, supernatant is abandoned, obtains bacterial sediment,
Precipitated 2 times with fresh LB culture mediums washing thalline, bacterial sediment is resuspended with 300 μ L LB fluid nutrient mediums, obtains recipient bacterium and hangs
Supernatant liquid；
(3) it is coated with after the acceptor bacterium suspension of the donor bacterium suspension of 30 μ L steps (1) and 300 μ L steps (2) is mixed
In on LB flat boards, 30 DEG C are cultivated 20-24 hours；
(4) bacterium grown on step (3) LB flat boards is eluted to EP pipes with the 1mL Sistrom culture mediums of precooling on ice
In.Centrifuged 2 minutes under 4 DEG C of 5000rpm, abandon supernatant, precipitation is washed twice with the Sistrom culture mediums of 500 μ l precoolings on ice,
Precipitation finally is resuspended with the Sistrom culture mediums of 100 μ L precoolings on ice, obtains thallus suspension liquid；
(5) by the thallus suspension liquid of step (4) be coated on Sistrom flat boards (Sistrom flat boards be to Sistrom cultivate
K is added in base2TeO3The K obtained with kanamycins2TeO3The solid training that concentration is 150mg/L, kanamycins concentration is 50mg/L
Support base) on, cultivated at 30 DEG C and grow within 3-5 days joint element, the restructuring hydrogenlike silicon ion as containing pBBR1MCS-2-Ts1, by this
Recombinant bacterium is named as R.s (pBBR1MCS-2).
According to the method for above-mentioned steps (1)-(5), recombination bacillus coli S17-1 (pBBR1MCS-2) is replaced with respectively
S17-1 (pBBR1MCS-2-Ts1), S17-1 (pBBR1MCS-2-Ts2), S17-1 (pBBR1MCS-2-Ts3) and S17-1
(pBBR1MCS-2-Ts4) it is, constant for his step, obtain respectively containing pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2,
PBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 restructuring hydrogenlike silicon ion, these recombinant bacteriums are respectively designated as R.s
(pBBR1MCS-2-Ts1), R.s (pBBR1MCS-2-Ts2), R.s (pBBR1MCS-2-Ts3) and R.s (pBBR1MCS-2-
Ts4)。
2nd, pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are in class ball
Temperature sensitivity in red bacterium Rhodobacter sphaeroides 2.4.1
The R.s (pBBR1MCS-2-Ts1) of step 1 is seeded in LB+kan fluid nutrient mediums, it is small in 30 DEG C of cultures 20
When, obtained bacterium solution is subjected to following handle：
1. bacterium solution is pressed 1：500 ratio is seeded in 3mL LB fluid nutrient mediums, is then cultivated 24 hours at 30 DEG C,
Obtain LB-30 DEG C of bacterium solution；LB-30 DEG C of dilution bacterium solution is obtained after LB-30 DEG C of bacterium solution is diluted, by LB-30 DEG C of dilution bacterium solution difference
It is applied on LB flat boards and LB+kan flat boards, each μ L LB-30 DEG C of flat board 100 dilute bacterium solution, are then cultivated 4 days at 30 DEG C,
LB-LB-30 DEG C of flat board and LB-LB+kan-30 DEG C of flat board are respectively obtained, counts LB-LB-30 DEG C of flat board and LB-LB+kan-30 DEG C
Clump count on flat board, Loss Rates of the recombinant vector pBBR1MCS-2-Ts1 in 30 DEG C of LB fluid nutrient mediums is calculated, carries
The Loss Rate of body=(clump count on-LB-LB+kan-30 DEG C of flat boards of clump count on LB-LB-30 DEG C of flat board) ÷ LB-LB-
Clump count on 30 DEG C of flat boards；
2. bacterium solution is pressed 1：500 ratio is seeded in 3mL LB+kan fluid nutrient mediums, then cultivates 24 at 30 DEG C
Hour, obtain LB+kan-30 DEG C of bacterium solution；LB+kan-30 DEG C of dilution bacterium solution is obtained after LB+kan-30 DEG C of bacterium solution is diluted, by LB+
Kan-30 DEG C of dilution bacterium solution is respectively coated to LB flat boards and LB+kan flat boards, each μ L LB+kan-30 DEG C of flat board 100 dilutions
Bacterium solution, then cultivated 4 days at 30 DEG C, respectively obtain LB+kan-LB-30 DEG C of flat board and LB+kan-LB+kan-30 DEG C of flat board,
The clump count on LB+kan-LB-30 DEG C of flat board and LB+kan-LB+kan-30 DEG C of flat board is counted, recombinant vector is calculated
Loss Rates of the pBBR1MCS-2-Ts1 in 30 DEG C of LB+kan fluid nutrient mediums, the Loss Rate of carrier=(LB+kan-LB-30 DEG C
The clump count on-LB+kan-LB+kan-30 DEG C of flat boards of clump count on flat board) bacterium colony on ÷ LB+kan-LB-30 DEG C flat boards
Number；
3. bacterium solution is pressed 1：500 ratio is seeded in 3mL LB fluid nutrient mediums, is then cultivated 24 hours at 37 DEG C,
Obtain LB-37 DEG C of bacterium solution；LB-37 DEG C of dilution bacterium solution is obtained after LB-37 DEG C of bacterium solution is diluted, by LB-37 DEG C of dilution bacterium solution difference
It is applied on LB flat boards and LB+kan flat boards, each μ L LB-37 DEG C of flat board 100 dilute bacterium solution, are then cultivated 4 days at 37 DEG C,
LB-LB-37 DEG C of flat board and LB-LB+kan-37 DEG C of flat board are respectively obtained, counts LB-LB-37 DEG C of flat board and LB-LB+kan-37 DEG C
Clump count on flat board, Loss Rate (tables of the recombinant vector pBBR1MCS-2-Ts1 in 37 DEG C of LB fluid nutrient mediums is calculated
6), the Loss Rate of carrier=(clump count on-LB-LB+kan-37 DEG C of flat boards of clump count on LB-LB-37 DEG C of flat board) ÷
Clump count on LB-LB-37 DEG C of flat board.
According to the method described above, R.s (pBBR1MCS-2-Ts1) is replaced with into R.s (pBBR1MCS-2-Ts2), R.s
(pBBR1MCS-2-Ts3), R.s (pBBR1MCS-2-Ts4) and R.s (pBBR1MCS-2), other steps are constant, respectively obtain
Recombinant vector pBBR1MCS-2, pBBR1MCS- when Host Strains are hydrogenlike silicon ion Rhodobacter sphaeroides 2.4.1
2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are respectively 30 DEG C of LB fluid nutrient mediums, 30
Loss Rate (table 6) in DEG C LB+kan fluid nutrient mediums and 37 DEG C of LB fluid nutrient mediums.
The Loss Rate of table 6, responsive to temperature type carrier in hydrogenlike silicon ion Rhodobacter sphaeroides 2.4.1
When Host Strains are hydrogenlike silicon ion Rhodobacter sphaeroides 2.4.1, pBBR1MCS-2,
PBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are in 30 DEG C of LB Liquid Cultures
The Loss Rate of Loss Rate and pBBR1MCS-2 in 37 DEG C of LB fluid nutrient mediums in base and 30 DEG C of LB+kan fluid nutrient mediums
Below 13%, pBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are 37
Loss Rate in DEG C LB fluid nutrient mediums respectively reaches 78.18%, 81.36%, 80.53% and 78.57%, explanation
PBBR1MCS-2-Ts1, pBBR1MCS-2-Ts2, pBBR1MCS-2-Ts3 and pBBR1MCS-2-Ts4 are in hydrogenlike silicon ion
Its certain temperature sensitivity can be shown in Rhodobacter sphaeroides 2.4.1.
Claims (4)
- It is that the DNA fragmentation shown in SEQ ID No.1 1557-2526 positions is replaced with into mesh 1. building the method for recombinant vector Expression casette, and keep SEQ ID No.1 other invariant nucleotides, obtained recombinant DNA is the recombinant vector；The protein of destination gene expression box coding is that the protein shown in SEQ ID No.2 is carried out into A2) transformation, protect Hold the SEQ ID No.2 constant obtained protein of other amino acid residues：The A2) it is by SEQ IDNo.2 the 130th His sport Gln；Target gene in the destination gene expression box is by shown in SEQ ID No.1 1794-2456 positions nucleotides DNA molecular carries out B2) transformation, keep its in DNA molecular shown in SEQ ID No.1 1794-2456 positions nucleotides The constant obtained DNA molecular of its nucleotides；The B2) it is that SEQ ID No.1 the 2183rd C is sported into G；The promoter for starting the destination gene expression in the destination gene expression box is by SEQ ID No.1 1557- DNA molecular shown in 1793 carries out following C2) transformation, keep shown in SEQ ID No.1 1557-1793 positions nucleotides DNA molecular in the constant obtained DNA molecular of other nucleotides：C2) following C21) and C22)：C21 SEQ ID No.1 the 1732nd A) is sported into G；C22 SEQ ID No.1 the 1780th G) is sported into T；The terminator that the destination gene expression is terminated in the destination gene expression box is SEQ ID No.1 2457- DNA molecular shown in 2526.
- 2. the recombinant vector built by claim 1 methods described.
- 3. recombinant microorganism or recombinant cell lines containing recombinant vector described in claim 2.
- 4. application of the recombinant vector described in claim 2 in as temperature sensitive vector.
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Citations (2)
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| WO2003076452A2 (en) * | 2002-03-08 | 2003-09-18 | E.I. Du Pont De Nemours And Company | Temperature sensitive mutant derivatives of the broad host range plasmid pbhr1 |
| CN103789337A (en) * | 2014-02-18 | 2014-05-14 | 扬州大学 | Suicide vector pGMB152 for blue-white selection and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003076452A2 (en) * | 2002-03-08 | 2003-09-18 | E.I. Du Pont De Nemours And Company | Temperature sensitive mutant derivatives of the broad host range plasmid pbhr1 |
| CN103789337A (en) * | 2014-02-18 | 2014-05-14 | 扬州大学 | Suicide vector pGMB152 for blue-white selection and application thereof |
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| Title |
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| Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants;DENISE K. GAVIN et al;《JOURNAL OF VIROLOGY》;19991130;p.9433–9445 * |
| Improvement of pBBR1MCS plasmids, a very useful series of broad-host-range cloning vectors;Sonja Obranic et al.;《Plasmid》;20131231;263–267 * |
| 类球红细菌遗传操作体系的构建及其产辅酶Q10代谢工程研究;周芬;《中国优秀硕士学位论文全文数据库 基础科学辑》;20160815;第四章 * |
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