CN103525806B - A kind of Streptomycete forward mutation strain screening method - Google Patents
A kind of Streptomycete forward mutation strain screening method Download PDFInfo
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- CN103525806B CN103525806B CN201310540710.9A CN201310540710A CN103525806B CN 103525806 B CN103525806 B CN 103525806B CN 201310540710 A CN201310540710 A CN 201310540710A CN 103525806 B CN103525806 B CN 103525806B
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Abstract
The present invention relates to microorganism strains screening field, particularly a kind of Streptomycete forward mutation strain screening method, comprises the following steps: strain mutagenesis; Bacterial strain that is original and mutagenesis is inoculated on regenerated plate respectively, and is cultured to growth and maturity; Being prepared into concentration is respectively 10
6-10
7the spore suspension of individual/ml; Take out the spore suspension of same volume, and the volume taken out is less than the cumulative volume of spore suspension; The spore suspension of taking-up is inoculated in respectively screen dull and stereotyped different plate hole substratum on and make it be uniformly distributed on substratum, be cultured to growth and maturity; From each plate hole, all take out the culture block of same volume, carry out lixiviate respectively and measure antibiotic content; The antibiotic content of mutagenic strain and original strain is contrasted, picks out forward mutation bacterial strain.Streptomycete forward mutation strain screening method provided by the invention, can avoid causing the bacterial strain filtered out to occur false positive because of the greatest differences of inoculum size, thus avoids screening the low problem of accuracy.
Description
Technical field
The present invention relates to microorganism strains screening field, in particular to a kind of Streptomycete forward mutation strain screening method.
Background technology
Existing microbiotic major part is produced by streptomycete.For improving the output of production of antibiotics bacterial strain, often needing to carry out mutagenesis to production bacterial strain, improving the antibiotic yield of this bacterial strain.But there is larger difficulty in the superior strain accurately picking out forward mutation from mass mutation strain.
At present, the screening method of superior strain mainly with the toothpick after sterilizing or transfering loop from picking list bacterium colony spore regenerated plate, spore is directly inoculated in is equipped with on 96 orifice plates of solid medium afterwards.Because spore inoculating amount exists greatest differences, the bacterium number grown in each plate hole of 96 orifice plate on substratum is different, and the substratum rounded afterwards in a plate hole carries out lixiviate, measures the antibiotic content produced and has very big-difference.Therefore, adopt toothpick or transfering loop picking regeneration bacterium colony spore directly to inoculate, being difficult to realize inoculum size consistent, easily there is false positive in the bacterial strain of screening.Therefore, there is the low problem of accuracy in above-mentioned screening method.
Summary of the invention
The object of the present invention is to provide a kind of Streptomycete forward mutation strain screening method, to solve the above problems.
A kind of Streptomycete forward mutation strain screening method provided in an embodiment of the present invention, comprises the following steps:
Mutagenesis is carried out to a part of original strain;
The bacterial strain of original strain and mutagenesis is inoculated on regenerated plate respectively, and is cultured to growth and maturity;
Get the original strain spore of growth and maturity and the mutagenic strain spore of multiple growth and maturity respectively, and to be prepared into concentration be respectively 10
6-10
7the spore suspension of individual/ml;
By the spore suspension taking out same volume in each spore suspension, and the volume taken out is less than the cumulative volume of spore suspension;
The spore suspension of taking-up is inoculated in respectively screen dull and stereotyped different plate hole substratum on and make spore suspension be uniformly distributed on substratum, be cultured to growth and maturity;
From each plate hole that screening is dull and stereotyped, all take out the culture block of same volume, carry out lixiviate respectively and measure antibiotic content;
The antibiotic content of the antibiotic content of mutagenic strain and original strain is contrasted, selects and produce the forward mutation bacterial strain of microbiotic ability higher than original strain.
In Streptomycete forward mutation strain screening method provided by the invention, got spore is made spore suspension and the concentration of spore suspension miospore is 10
6-10
7individual/ml, defines the concentration difference of spore suspension prepared by different strains.The concentration of suspension miospore is relatively homogeneous, same volume by the spore suspension taken out in each spore suspension, and the volume taken out is less than the cumulative volume of spore suspension, therefore, spore quantity for inoculating is less than total spore quantity, and like this for each bacterial strain, the difference between the spore quantity of inoculation reduces, so just can avoid causing the bacterial strain filtered out to occur false positive because of the greatest differences of inoculum size, thus avoid screening the low problem of accuracy.
Accompanying drawing explanation
Fig. 1 to show when adopting liquid culture medium filling the disparity map of substratum weight in each plate hole under differing temps;
Fig. 2 shows the disparity map of substratum weight in each plate hole when adopting culture block filling;
Fig. 3 shows ultrasonic oscillation extraction efficiency figure.
Embodiment
Also by reference to the accompanying drawings the present invention is described in further detail below by specific embodiment.
Embodiment 1:
A kind of Streptomycete forward mutation strain screening method provided in an embodiment of the present invention, comprises the following steps:
Mutagenesis is carried out to a part of original strain;
The bacterial strain of original strain and mutagenesis is inoculated on regenerated plate respectively, and is cultured to growth and maturity;
Get the original strain spore of growth and maturity and the mutagenic strain spore of multiple growth and maturity respectively, and to be prepared into concentration be respectively 10
6-10
7the spore suspension of individual/ml;
By the spore suspension taking out same volume in each described spore suspension, and the volume taken out is less than the cumulative volume of spore suspension;
The spore suspension of taking-up is inoculated in respectively screen dull and stereotyped different plate hole substratum on and spore suspension is uniformly distributed on described substratum, be cultured to growth and maturity;
From each plate hole that screening is dull and stereotyped, all take out the culture block of same volume, carry out lixiviate respectively and measure antibiotic content;
The antibiotic content of the antibiotic content of mutagenic strain and original strain is contrasted, selects and produce the forward mutation bacterial strain of microbiotic ability higher than original strain.
The toothpick of employing sterilizing or transfering loop are from picking list bacterium colony spore regenerated plate, directly carry out inoculation screening, because spore inoculating amount exists greatest differences, the bacterium number of growth in each plate hole of 96 orifice plate on substratum is different, the substratum rounded afterwards in a plate hole carries out lixiviate, measures the antibiotic content produced and has very big-difference.Therefore, the forward mutant picked out like this is incredible.In the Streptomycete forward mutation strain screening method that the present embodiment provides, got spore is made spore suspension and the concentration of spore suspension miospore is 10
6-10
7individual/ml, defines the concentration difference of spore suspension prepared by different strains.The concentration of suspension miospore is relatively homogeneous, same volume by the spore suspension taken out in each spore suspension, and the volume taken out is less than the cumulative volume of spore suspension, therefore, spore quantity for inoculating is less than total spore quantity, and like this for each bacterial strain, the difference between the spore quantity of inoculation reduces, so just can avoid causing the bacterial strain filtered out to occur false positive because of the greatest differences of inoculum size, thus avoid screening the low problem of accuracy.
Embodiment 2:
Based on embodiment 1, the present embodiment has done further improvement:
For the step preparing spore suspension in embodiment 1, can be obtained by the following step:
With the spore of original strain of growth and maturity and the spore of the mutagenic strain of multiple growth and maturity of the identical number of rings of transfering loop difference picking;
The spore of picking is added to respectively different from the aseptic culture presevation pipe of sterilized water and granulated glass sphere;
All culture presevation pipes are added a cover to be placed on vortex vortex mixer and shakes, spore chain is ruptured, obtained spore suspension.
Learn through counting, the spore adopting the transfering loop of 10 μ l specifications often to get 1 ring growth and maturity joins in 1.0ml sterilized water, and spore number is 10
6-10
7individual, namely the concentration of spore is 10
6-10
7individual/ml.Accordingly, the variable format of transfering loop, as 5 conventional μ l specifications; Accordingly, the number of rings of inoculation is also variable, and the number of rings of institute's picking is relevant with the spore amount of the specification of ring and each bacterial strain.Granulated glass sphere in preservation pipe on vortex vortex mixer gets up to contribute to smash spore chain with water flow rotary concussion, and therefore, accordingly, the specification of preservation pipe is variable, as long as corresponding to got liquid volume; Accordingly, the specification of granulated glass sphere and variable amounts, be as the criterion to smash spore chain.Preferably, adopting the spore 1-2 ring of the transfering loop picking growth and maturity of 10 μ l specifications to being in the 2ml plastics culture presevation pipe of 3mm granulated glass sphere containing 1.0ml sterilized water and 2-3 grain diameter, after vortex vortex mixer shaking 2min, obtaining spore suspension.In addition, spore growth maturation refers to that growth has the media surface of bacterial strain to occur flat-white or canescence.
In order to improve screening efficiency, ensure that the bacterial strain in each plate hole still leaves part spore for propagating after having measured antibiotic content, preferably, the diameter of plate hole is 6-20mm, and the degree of depth is 6-20mm simultaneously.
Dull and stereotyped 24 well culture plates commonly used for laboratory of 24 hole sizers choosings, specification is: plate length × plate is wide=128 × 86mm, bore dia 16mm, hole depth 16mm, and each hole is identical specification; Cultivate the culture plate of bacterium colony, be advisable with the solid medium containing 1/3-1/2 volume in culture plate.The results showed, comparatively suitable containing 1.0-1.5ml substratum in each plate hole that 24 hole sizer choosings are dull and stereotyped: add very little, after placing the long period, dry and cracked phenomenon easily appears in solid medium; Add and then cause plate hole superjacent air space too little too much, be unfavorable for storing a certain amount of air in plate hole, for the respiration in strain growth process.Therefore, preferably, the diameter of described plate hole is 16mm, and the degree of depth is 16mm, and containing 1.0-1.5ml substratum in each plate hole.
At present, the preparation of substratum is generally directly added by the liquid culture medium of heating and melting in culture vessel to obtain, but, for the present embodiment, need filling substratum in multiple plate hole, in the liquid culture medium process of same volume filling in plate hole, liquid culture medium temperature constantly declines, because the liquid culture medium density temperature influence of heating and melting is comparatively large, therefore, cause adding the weight fluctuation of substratum in plate hole larger.Dull and stereotyped for 24 hole sizers choosings, adopt liquid culture medium filling, the difference of substratum weight in each plate hole under determining differing temps, accordingly, take X-coordinate as plate hole number, ordinate zou is that the weight of substratum in each plate hole obtains Fig. 1.As shown in Figure 1, when when 85 DEG C filling, in each plate hole, the relative standard deviation of culture block weight can reach 15.02%, and extreme difference value (maxima and minima is poor) can reach 823mg; When 50 DEG C filling, in each plate hole, the relative standard deviation of culture block weight can reach 7.91%, and extreme difference value can reach 554.9mg.For this reason, substratum packaging process is improved, particularly, pouring process is as follows: in 9cm culture dish, add the liquid culture medium of 50-60ml through heating and melting, after liquid culture medium solidifies, beat from culture dish with diameter 15mm punch tool and get culture block, proceeded in the plate hole of 24 orifice plates, in each plate hole, add one piece of culture block; Adding a cover post-heating makes culture block in plate hole melt, for subsequent use after liquid culture medium cooled and solidified in plate hole.Afterwards the substratum in above-mentioned each plate hole is measured weight, take X-coordinate as plate hole number, ordinate zou is that the weight of substratum in each plate hole obtains Fig. 2.As shown in Figure 2, in each plate hole, substratum standard weight deviations controls, in 2.5% scope, to effectively reduce the weight differential of substratum between plate hole.Adopt solid agar block encapsulating method to replace conventional liquid encapsulating method, ensure that in each plate hole, substratum actual content height is consistent, improves the preci-sion and accuracy of screening model.Preferably, the preparation method of the substratum of plate hole comprises the following steps: the substratum getting same volume from same solid medium, puts into each plate hole respectively; Plate hole is added a cover and carries out heating and make it melt rear cooled and solidified.
To get spore suspension volume be determine according to the dry wet degree of cultivating the size of plate hole and solid culture primary surface.Get too many suspension can cause in plate hole and occur ponding, cause anoxic condition, be unfavorable for that spore germination grows; Get very little suspension can due to liquid very little, spore suspension cannot be uniformly distributed in media surface in plate hole.The results showed, get in the dull and stereotyped each plate hole of spore suspension 20-50 μ l to 24 hole sizer choosing and be advisable, rock 24 hole sizer choosing flat boards gently, spore suspension is uniformly distributed on the surface of substratum in plate hole.
The bacterial strain of mutagenesis and original strain are inoculated on regenerated plate respectively, and be cultured in the step of growth and maturity, 7 days are needed to growth and maturity after inoculation, in addition, the strain culturing measured also needs a couple of days to growth and maturity and the antibiotic process of mensuration, therefore, after completing etc. mensuration, return regenerated plate again and get on again to select bacterial strain spore, the regenerated plate substratum be kept in biochemical cultivation case is often dry and cracked owing to placing for a long time, be difficult to picking out spore from above, this step very easily causes the dominant strain screened to lose.In order to address this problem, preferably, the culture block of taking-up is less than the diameter of plate hole, and like this, spore residual in plate hole can be used for the preservation of bacterial strain.
Punch tool is a kind of common instrument, and for beating, to get culture block simple and easy to do, and therefore, preferably, culture block is beaten by punch tool and got from plate hole.
Prove through test, the dull and stereotyped upper inoculating spores of 24 hole sizer choosing, growth and maturity after 4 days, the bacterial strain caused to prevent the reasons such as substratum is dry and cracked is dead, there are enough available spores for preserving bacterial strain, select diameter to be that the culture block of getting in the dull and stereotyped upper plate hole of 24 hole sizers choosing beaten by the punch tool of 5mm, preferably, the diameter of punch tool is 5mm; Beat the volume of the culture block of getting in the dull and stereotyped upper plate hole of 24 hole sizer choosing according to the punch tool of 5mm, preferably, the capacity of pipe is 2ml; The vat liquor anhydrous methanol added is advisable with submerged culture matrix, and empirical tests is learnt, 0.6ml and above volume get final product submergence, preferably, selects 1ml anhydrous methanol; Due to anhydrous methanol highly volatile, identical in order to ensure the anhydrous methanol volume in each plastics tubing, should add a cover immediately after anhydrous methanol adds plastics tubing and carry out ultrasonic oscillation; Material in pipe after concussion is obtained vat liquor after filtering with microporous membrane.Preferably, vat liquor adopts liquid chromatogram measuring antibiotic content.
The 2ml containing culture block and 1ml anhydrous methanol obtained by aforesaid method manages, lixiviate different time is shaken under being placed in ultrasonic wave, material in pipe after concussion is obtained vat liquor after the filtering with microporous membrane of 0.45 μm, through liquid chromatogram measuring antibiotic content, obtain experimental data as table 1, with the ultrasonic time of table 1 for X-coordinate, mensuration peak height is ordinate zou, can obtain ultrasonic oscillation extraction efficiency as shown in Figure 3.
Table 1: ultrasonic oscillation extraction efficiency table
| Ultrasonic time (min) | 5 | 10 | 15 | 20 | 25 | 30 |
| Measure peak height (mAU) | 10.27 | 22.09 | 24.46 | 25.03 | 24.97 | 25.13 |
Can demonstrate antibiotic relative content by measuring peak height, as can be seen from table 1 and Fig. 3, under normal temperature, ultrasonic oscillation lixiviate 20min reaches between stationary phase, and therefore, the time of ultrasonic oscillation is preferably 20min.
In pipe after lixiviate, material obtains vat liquor after filtering, filters the method adopted and carries out according to follow-up test.If vat liquor adopts liquid chromatogram measuring antibiotic content, in order to prevent the agar pieces in substratum and the spore that splits away off from causing liquid chromatography to block, general liquid chromatogram mobile phase is all through the membrane filtration of 0.45 μm.So select the membrane filtration of 0.45 μm in test, but also can adopt the membrane filtration of thinner specification filtration as 0.20 μm.
In addition, in order to make streptomycete bacterial strain well-grown, preferably, the composition of regenerated plate substratum is: glucose: 18g/L, peptone 5g/L, dregs of beans 3g/L, yeast extract paste 1g/L, CaCO
30.25g/L, agar powder 2.0g/L; PH=8.0-8.5.Preferably, screening culture medium can be selected to add corresponding selective agent on the basis of regenerated plate substratum.
According to the method for the present embodiment, same bacterial strain is adopted to mode Simultaneous vaccination 10 plate holes of spore suspension, antibiotic content fluctuation between each plate hole can be controlled within 30%, and adopts direct inoculation, and between 10 plate holes, antibiotic content difference can reach 300%-500%.Visible, the spore suspension method inoculation of employing more can the true antibiotic yield of this bacterial strain of direct reaction.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. a Streptomycete forward mutation strain screening method, is characterized in that, comprises the following steps:
Mutagenesis is carried out to a part of original strain;
The bacterial strain of original strain and mutagenesis is inoculated on regenerated plate respectively, and is cultured to growth and maturity;
Get the original strain spore of growth and maturity and the mutagenic strain spore of multiple growth and maturity respectively, and to be prepared into concentration be respectively 10
6-10
7the spore suspension of individual/ml;
By the spore suspension taking out same volume in each described spore suspension, and the volume taken out is less than the cumulative volume of spore suspension;
The spore suspension of taking-up is inoculated in respectively screen dull and stereotyped different plate hole substratum on and spore suspension is uniformly distributed on described substratum, be cultured to growth and maturity, the preparation method of the substratum of described plate hole comprises the following steps:
Get the substratum of same volume from same solid medium, put into each plate hole respectively;
Plate hole is added a cover and carries out heating and make it melt rear cooled and solidified;
From each plate hole that described screening is dull and stereotyped, all take out the culture block of same volume, carry out lixiviate respectively and measure antibiotic content, described culture block is less than the diameter of described plate hole;
The antibiotic content of the antibiotic content of mutagenic strain and original strain is contrasted, selects and produce the forward mutation bacterial strain of microbiotic ability higher than original strain.
2. a kind of Streptomycete forward mutation strain screening method according to claim 1, is characterized in that, gets the original strain spore of growth and maturity and the mutagenic strain spore of multiple growth and maturity respectively described, and to be prepared into concentration be respectively 10
6-10
7in the step of the spore suspension of individual/ml, described spore suspension is specifically obtained by the following step:
With the spore of original strain of growth and maturity and the spore of the mutagenic strain of multiple growth and maturity of the identical number of rings of transfering loop difference picking;
The described spore of picking is added to respectively different from the aseptic culture presevation pipe of sterilized water and granulated glass sphere;
All described culture presevation pipes are added a cover to be placed on vortex vortex mixer and shakes, spore chain is ruptured, obtained spore suspension.
3. a kind of Streptomycete forward mutation strain screening method according to claim 1, it is characterized in that, described the spore suspension of taking-up is inoculated in respectively screen dull and stereotyped different plate hole substratum on and spore suspension is uniformly distributed on described substratum, be cultured in the step of growth and maturity:
The diameter of described plate hole is 6-20mm, and the degree of depth is 6-20mm.
4. a kind of Streptomycete forward mutation strain screening method according to claim 3, it is characterized in that, described the spore suspension of taking-up is inoculated in respectively screen dull and stereotyped different plate hole substratum on and spore suspension is uniformly distributed on described substratum, be cultured in the step of growth and maturity:
The diameter of described plate hole is 16mm, and the degree of depth is 16mm, and containing 1.0-1.5ml substratum in each plate hole.
5. a kind of Streptomycete forward mutation strain screening method according to claim 4, is characterized in that, described by each described spore suspension in take out the spore suspension of same volume, and the volume taken out is less than in the step of the cumulative volume of spore suspension:
Be 20-50 μ l by the volume taken out in each described spore suspension.
6. a kind of Streptomycete forward mutation strain screening method according to claim 5, is characterized in that, all takes out the culture block of same volume, carry out lixiviate respectively and measure in the step of antibiotic content described each plate hole dull and stereotyped from described screening:
Described culture block is beaten by punch tool and is got from described plate hole.
7. a kind of Streptomycete forward mutation strain screening method according to claim 6, it is characterized in that, the described each plate hole dull and stereotyped from described screening, all take out the culture block of same volume, carry out lixiviate respectively and measure in the step of antibiotic content, the diameter of described punch tool is 5mm; Described lixiviate specifically comprises the following steps:
Described culture block being shifted into capacity is in the pipe of 2ml, adds the anhydrous methanol being no less than 0.6ml, adds a cover immediately and carries out ultrasonic oscillation;
Material in pipe after concussion is obtained vat liquor after filtering with microporous membrane.
8. a kind of Streptomycete forward mutation strain screening method according to claim 7, is characterized in that, in described lixiviate step:
The add-on of described anhydrous methanol is 1ml;
The time of described ultrasonic oscillation is 20min.
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| CN102268378A (en) * | 2011-07-14 | 2011-12-07 | 华东理工大学 | Method for screening high yield strains from aerobic bacteria at high flux |
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| CN102268378A (en) * | 2011-07-14 | 2011-12-07 | 华东理工大学 | Method for screening high yield strains from aerobic bacteria at high flux |
Non-Patent Citations (2)
| Title |
|---|
| A high-throughput method for screening of rapamycin-producing strains of Streptomyces hygroscopicus by cultivation in 96-well microtiter plates;Xu et al;《Biotechnology Letters》;20051231;第1136页左栏第2-3段，图1 * |
| 阿维菌素B1a 组分高产菌株的高通量选育;宗莎莎等;《广东农业科学》;20111231;148-152 * |
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