CN106894094A - The King George Island, antarctica soil metagenome library for building - Google Patents
The King George Island, antarctica soil metagenome library for building Download PDFInfo
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- CN106894094A CN106894094A CN201510947454.4A CN201510947454A CN106894094A CN 106894094 A CN106894094 A CN 106894094A CN 201510947454 A CN201510947454 A CN 201510947454A CN 106894094 A CN106894094 A CN 106894094A
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
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Abstract
The present invention relates to fields such as edaphon and molecular biology, the large fragment DNA of edaphon about 40kb is especially isolated and purified out from King George Island, antarctica soil, be connected in suitable carrier and build grand genomic library.Include protokaryon and the various chromosome internal gene information of eukaryotic microorganisms in King George Island, antarctica soil in the library.The present invention is for the redundant gene resource contained in the soil of the South Pole, separate microorganism large fragment DNA and it is connected in the middle of grand genome vector, the various microbial resources in South Pole ecological environment of soil has been directly obtained in the state of not cultivating from the soil of the South Pole using Modern Molecular Biotechnology method.The library can be used to screen various different South Pole resources of soil microorganisms genes etc..
Description
Technical field
The present invention relates to fields such as extreme environment environmental microorganism, biochemistry and molecular biology, particularly from south
The DNA that the kb large fragments of edaphon about 40 are isolated and purified out in the King George Island soil of pole is connected to structure in Fosmid carriers
Into grand genomic library, include in the library in the soil of the South Pole protokaryon and the various chromosome internal genes of eukaryotic microorganisms and its
Regulation and controlling of information.
The present invention is directed to a large amount of biomass based productions contained in the soil of the South Pole, using molecular biotechnology means from the South Pole
Separate microorganism large fragment DNA and it is connected in the middle of grand genome vector in soil, is directly obtained in the case where not cultivating
Various microbial gene resources in the ecological environment of soil of the South Pole.The library can be used to analyze South Pole soil microbial community diversity
With the various novel microbes genetic resourceses of screening.
Background technology
According to current scientific circles it is widely recognized that microorganism only has 0.01 ~ 10% and can cultivate in nature perches group,
Due to lack reproducing environment condition method and suitable culture matrix, most of microorganism under conditions of current laboratory not
Or can be difficult manually to be cultivated, therefore its pure culture can not possibly be obtained and the characteristics such as form, physiology, heredity are carried out to it grind
Study carefully.Grand genome is the new term proposed for 1998 by Handelsman etc., and it is defined as " the genomes of the
Total microbiota round in nature ", i.e., the summation of whole tiny organism inhereditary materials in habitat.It is contained
The gene of educable and not educable microorganism, the genome that bacterium in environmental sample and fungi are referred mainly at present is total
With.Up to the present, soil metagenome technology is in gene identification, the discovery of bioactivator and soil ecology
It is widely used etc. several aspects.The main work(that newly encoded gene is the technology is obtained from soil metagenome library
Can where, it has been found that new gene mainly have biocatalyst gene, antibiotics resistance gene and coding transporter gene.
The most noticeable contribution of soil metagenome technology is the discovery of neoformation catalyst, including amylase, lipase, ester
Enzyme, protease, oxidoreducing enzyme etc., and many features information of new enzyme is obtained on this basis.
Now with the development of modern molecular biology, the need for biological DNA molecular level research, genome is built
Library becomes the conventional research method in laboratory.It can separate specific genetic fragment, study expression of gene etc., for
The research of the mankind and animal-plant gene group has very important effect.The carrier for building genomic library is a lot, can substantially divide
It is panel（Such as bacteriophage of early stage, Cosmid, PI bacteriophage and Fosmid）With artificially colored series（Such as
YAC, BAC and PAC etc.）.The former cloning capacity is relatively small（35~45kb of bacteriophage 24kb or so, Cosmid）,
And a series of artificial chromosomes for building, such as yeast artificial chromosome（Yeast artificial chromosome, YAC）、
Bacterial artificial chromosome（Bacterial artificial chromosome, BAC）, P1 artificial chromosomes（PI-derived
Artificial chromosome, PAC）And artificial mammalian chromosome（mammalian artificial
Chromosome, MAC）The gene structure huge and complicated etc. disclosure satisfy that eucaryote.Fosmid carriers are current trends
New support for substituting Cosmid vector construction large fragments library, compared with BAC library constructions, the structure in Fosmid libraries
It is simpler, quickly.In recent years, Fosmid libraries have been widely used in the map based cloning of gene, the structure of physical map and ratio
Compared with genome research.Its advantage is as follows.
1. due to inserting the Escherichia coli F factor F- factors, so it exists in Host Strains in single copy form,
Good stability.
2. have a derivable oriV copy replication origin high on Fosmid carriers, it is necessary to when inducible reach height
Copy (50 or so).
3. randomness is good, it is ensured that the frequency occurred in library per segment DNA is impartial.
The unique natural environment in the South Pole causes that this area is richly stored with microbial resources, and contains various important
Novel microbes.Situations such as domestic and international researcher is to the distribution of polar microorganism is made that substantial amounts of research, it is found that the South Pole is micro-
Biological species is very more, and many secondary metabolites that this area microorganism produces are owned by novel bioactivity simultaneously
With the potential for being used as new medicine.Additionally, people are also isolated to many microbial species from polar region.Due to this
In the slightly biological long-term environment for living in cold, they form various different adaptations when these bitter cold environment are adapted to should
The metabolic mechanism of environment, then just makes these microorganisms and the metabolite produced by them be provided with certain uniqueness,
Contain immeasurable application potential.
The present invention aiming at a large amount of microbial gene group informations contained in the soil of the South Pole, using soil large fragment DNA
Extractive technique and Fosmid carriers, have been directly obtained including non-culture microbial gene by way of building grand genomic library
Group information is in interior all of geobiont information.The structure in the library can be satisfied with the separation of various enzyme genes, antibiotic
Screening and molecular biology research need.
The content of the invention
Fosmid carriers are the new supports for substituting Cosmid vector construction large fragments library of current trend, with
BAC library constructions are compared, and the structure in Fosmid libraries is simpler, quickly.The carrier that the present invention is used according to practical operation experience
It is pCC2FOS, the soil large fragment of recovery is the fragment in 40kb or so sizes, and Host Strains are Escherichia coli.Present invention success
Construct different loci South Pole soil metagenome Fosmid libraries, while point out each build committed step operation in note
The use scope in meaning item and the library etc. is actually needed reference for everybody according to oneself.
For the extraction and recovery of soil large fragment DNA, the method that we use kit, the large fragment of extraction
DNA meets following requirement simultaneously：1. fragment will about in 40kb, because fragment is big constructed in identical clone's number
The information content that library includes is just big, could enough contents for covering hereditary information in soil；2. purity is high, is especially free of
Or it is few containing the material for suppressing enzyme activity in follow-up experiment (this kind of material in soil is a lot)；3. to the greatest extent may be used in the DNA for reclaiming
The hereditary information comprising all microorganisms in soil of energy.
The invention provides a complete South Pole soil metagenome Fosmid library comprising the grand above of 10 myriagrams.
The invention provides the points for attention and principle of each committed step of soil metagenome library construction, can piece as needed
Duan great little selects different types of carrier and construction step.
The invention provides the principle and method that can according to actual needs change operating procedure.
Specifically, there is provided whole process of the library from large fragment soil DNA to library clone.
The invention provides the detailed step and principle of each key link, and propose can with the suggestion of Adjusted Option and
Method.Having pointed out initially that will obtain than larger DNA fragmentation, and DNA fragmentation is more than 25kb, can otherwise cause strange clone.Secondly
The points for attention in connection procedure are indicated, the amount of the suitable plasmid needed for calculating first required for the library of structure.One
Individual reaction will produce 103-106Individual clone, is specifically dependent upon the quality of " insertion DNA ".Can be calculated by these needs connection
The number of times of reaction, coupled reaction can expand and diminution system.
The library that the present invention is provided can be used for the screening of industrial enzymes gene, the discovery of antibiotics, agricultural with poisonous
The research work of the degrading genes of harmful substance, the regulation and controlling of information of microbial gene etc., it is also possible to meet the follow-up of some correlations
Need of work.Specifically, the invention provides the structure cube method and the text for having very much development and application researching value of acquisition in library
Storehouse, specific method is as follows.
1., disclosure sets forth the construction method step in the library, according to actual operation and disclosed side is combined
Method has put into practice out practicable construction method, and obtains a capacity grand base of soil above grand more than 10 myriagrams
Because of a group library.
2. the DNA molecules of different size fragment and the connection of carrier, calculate first build library required for it is suitable
Plasmid amount depend on ' insertion DNA ' quality, can calculate the number of times for needing coupled reaction, coupled reaction by these
Can expand and diminution system.
3. the packaging of Fosmid clones, connection product is packed, the phages infection EPI300- after dilution
T1R Host Strains.
According to the principle of present invention offer and by implementing specific method of the invention and combining to need to adjust operating procedure,
Following Expected Results can be reached.
1. different loci South Pole soil metagenome library is provided, and the library of acquisition can be used for various work(
Can the screening of gene, the separation of microbial secondary metabolite and various follow-up molecular biology etc. scientific research.
2., the invention provides the construction method in the soil metagenome library for obtaining, have compared with conventional method detailed
Carefully, the features such as workable, operating procedure is according to needing adjustable.
Specific embodiment
Below, the present invention is illustrated for embodiment, but, the present invention is not limited to following embodiments.
The soil DNA extraction purification of embodiment one
South Pole soil STb gene is extracted using Mobio PowerSoil DNA Isolation Kit, step is as follows：
1st, take in 0.25g pedotheques addition PowerBead pipes, be gently vortexed and mix；
2nd, 60 μ L Solution C1 are added, is turned upside down and mix for several times；
4th, PowerBead pipes are fixed on vortex instrument adapter, maximum (top) speed 3200rpm vortex continuous oscillations 10min；
5th, room temperature 10000g centrifugations 30s, in transfer supernatant to a clean 2mL collecting pipe；
6th, 250 μ L Solution C2 are added to mix 5s in supernatant, being vortexed, 4 DEG C of incubation 5min；
7th, room temperature 10000g centrifugations 1min, avoids precipitating globule, in transfer supernatant to a new collecting pipe；
8th, add 200 μ l Solution C3 in supernatant, be vortexed and mix, 4 DEG C of incubation 5min；
9th, room temperature 10000g centrifugations 1min, avoids precipitating globule, in transfer supernatant to a new collecting pipe；
10th, add 1200 μ L Solution C4 in supernatant, be vortexed and mix 5s；
11st, about 675 μ L of supernatant are loaded in Spin Filter, room temperature 10000g centrifugation 1min discard filtrate, continue to load 675
μ L of supernatant, room temperature 10000g centrifugation 1min, is repeated up to filter all supernatants；
12nd, add in 500 μ L Solution C5 to Spin Filter, room temperature 10000g is centrifuged 30s, supernatant discarded；
13rd, room temperature 10000g centrifugations 1min, in careful transfer Spin filter to 2mL collecting pipes；
14th, 100 μ L Solution C6 to white filter membrane center, room temperature 10000g centrifugations 30s are added；
15th, Spin Filter are discarded, now the DNA in collecting pipe can be directly used for downstream experiment, without being further purified.
The connection of the large fragment of embodiment two and carrier.
1.Gelase digestion recovery has large fragment DNA sample blob of viscose, and the concentration of large fragment DNA is detected after the completion of digestion.
2. mix following solvent, and it is every add a kind of reagent after thoroughly mix, the coupled reaction body of 10 μ L of composition
System.PCC2FOS and " insertion DNA " are 10 in mol ratio：It is most suitable when 1：
Sterile ddH2O 2μL
Fast-Link 10 x ligation Buffer 1μL
（10mmol/L）ATP 1μL
pCC2FOS Vector 1μL
Insertion DNA 4 μ L
Fast-Link DNA Ligase 1μL
Incubated at room temperature 4 hours, is transferred to reaction system 70 DEG C of water-baths and is incubated 10 minutes, makes Fast-Link DNA Ligase enzymes
Inactivation.
The packaging of the Fosmid of embodiment three clones, transfection
Packaging reaction the previous day, by the Host Strains EPI300-T1R Plating Strain of incubated overnight be inoculated into containing
In the 50mL LB culture mediums of 10mmol/L magnesium sulfate and 0.2% maltose, 37 DEG C of shaken cultivations to OD600Between 0.8-1.0
（About 2h）, 4 DEG C of preservations.
Packaging, transfection procedure are specific as follows.
1st, the pipe MaxPlax Lambda packaging Extracts of thawed on ice 1.
2nd, immediately in the aseptic EP pipes of the packaging Extracts to another 1.5mL of the μ L above-mentioned 1 of transferase 12 5, will be surplus
25 remaining μ L packaging Extracts put back to -70 DEG C of refrigerators.
3rd, 10 μ L coupled reaction systems in above-mentioned case study on implementation two are taken, is added in above-mentioned 2 aseptic EP pipes, use liquid-transfering gun
Liquid is mixed, notes avoiding producing bubble.400rpm low-speed centrifugals, collect solution to EP bottom of the tube.
4th, above-mentioned 3 reaction solution is packed after reacting in being packed 2 hours at 30 DEG C, remaining 25 μ L in adding above-mentioned 2
Packaging Extracts, mix.
5th, above-mentioned 4 reaction solution after packaging reaction, adds bacteriophage dilute in second packaging reaction 2 hours at 30 DEG C
Release buffer solution（Phage Dilution Buffer）Mix to 1.5mL and gently.According to every 100 μ L EPI300-T1R Host Strains
The ratio of phages mixes both after adding 10 μ L to dilute in solution, and 37 DEG C of phage adsorption 1h will infect phagocytosis
The bacterium of body is applied on the LB flat boards containing 12.5 μ g/mL chloramphenicol, and 37 DEG C are cultivated 12 ~ 16 hours, obtains conversion flat board.Or
Often pipe phages add 25 μ L chloroforms, 4 DEG C of preservations after gentle mixing to person.
The amplification in example IV library is preserved and detected.
1st, 1 ~ 2 ml LB fluid nutrient mediums are added to the conversion flat board of above-mentioned steps 5, suspension thalline cell；Transfer is outstanding
Floating cell is converted on flat board to another, suspension thalline cell；And repeat the step.
2nd, final bacteria suspension being shifted in aseptic EP pipes, adding sterile glycerol its final concentration is reached 20%, mixing is divided into
Aliquot is stored in 70 °C.
3rd, colony counting is carried out, Fosmid library phages titres is calculated according to below equation：
Phage titre=（Flat-plate bacterial colony number × 100 μ LmL-1× extension rate）/ coating volume.
4th, result：In above-mentioned 3, the phage titre in constructed Fosmid libraries is 1.12 × 106 cfu/mL.South Pole soil
The average length of the grand genome insertion DNA of earth is 40kb, is 5Mb by the mean size of the microbial genome in the soil of the South Pole
Calculate, then according to following formula, the microbial cell number that can be calculated in the sample of library covering is about 5.8 × 105It is individual,
Meet library construction standard.
Clone number=（Phage titre × packaging volume）/[ln(1-P)/ln(1-f)]
Wherein P=0.9999, is the desired value of covering measures.F=40kb/5Mb, is that the Insert Fragment in each Fosmid clones is long
Degree accounts for the fraction of average gene group；Packaging volume is 1ml.
Claims (4)
1. a South Pole soil metagenome library for structure, King George Island, antarctica soil is obtained using the method for molecular agents box
STb gene, then using 30-35V voltages, 10 hours electrophoretic separation all size DNA fragmentation, be finally recovered to 40kb or so
DNA fragmentation be connected and be cloned into e. coli host bacteria with Fosmid carriers, more than 100,000 clones, be built into one
Soil metagenome library.
2. a kind of all prokaryotic gene group relevant informations comprising the grand genomic library according to claim 1, it is special
Levy is containing a large amount of non-culture soil prokaryotic micro-organisms genomic informations in the library, wherein may be containing intersecting in each clone
The prokaryotic gene information of overlap.
3. a kind of all eukaryotic gene group relevant informations comprising the grand genomic library according to claim 1, it is special
Levy is containing a large amount of non-culture soil eukaryotic microorganisms genomic informations in the library, wherein may be containing intersecting in each clone
The eukaryotic gene biological information of overlap.
4. it is a kind of according to claim 1 comprising with the grand genomic library in gene and gene cluster regulation and controlling of information etc..
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| CN201510947454.4A CN106894094A (en) | 2015-12-17 | 2015-12-17 | The King George Island, antarctica soil metagenome library for building |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523562A (en) * | 2017-08-29 | 2017-12-29 | 海南省农业科学院植物保护研究所 | The anti-cucurbits fusarium wilt activated product screening in Fosmid libraries and authentication method |
| WO2019036899A1 (en) * | 2017-08-22 | 2019-02-28 | 深圳华大基因股份有限公司 | Optimized method for extracting large-fragment dna |
-
2015
- 2015-12-17 CN CN201510947454.4A patent/CN106894094A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019036899A1 (en) * | 2017-08-22 | 2019-02-28 | 深圳华大基因股份有限公司 | Optimized method for extracting large-fragment dna |
| CN107523562A (en) * | 2017-08-29 | 2017-12-29 | 海南省农业科学院植物保护研究所 | The anti-cucurbits fusarium wilt activated product screening in Fosmid libraries and authentication method |
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Application publication date: 20170627 |