CN112921050A - High-efficiency zero-background assembly method without homology and multiple long fragments - Google Patents
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Abstract
The invention discloses a method for efficiently forming a zero background group without homology and multiple long fragments, which comprises the following steps: firstly, adding a B fragment Yescaror I at the unique position of the Not I site of the whole circularization plasmid restriction endonuclease, simultaneously ensuring that the left and the right of the B fragment have just two Not I enzyme cutting sites, wherein two Pr imer1/2 primers are designed between A, B fragments without homology, and two Pr imer3/4 primers are designed between B, C fragments without homology and serve as bridges connected between the two fragments; transferring all the fragments into 7 ABCDEFGs in total and 7 pairs of pr imers 1-14 in total together with a pair of primers at the joint of each fragment into yeast competent cells for shake culture for two days; fourthly, extracting and culturing yeast plasmids for two days; finally, the method is obtained through amplification and detection, the zero background cloning is completed through yeast assembly and escherichia coli screening, no empty vector is produced, and compared with the traditional cloning mode, a large amount of time is saved.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a high-efficiency zero-background assembly method without homology and multiple long fragments.
Background
At present, because gene fragments have no homology, the 6 fragments can not be sequentially connected into a ring at one time, the conventional gene synthesis method is completed by segmenting and sequentially carrying out multiple steps, and more steps of a round of manager are needed.
Disclosure of Invention
The invention aims to provide a method for assembling a plurality of long fragments with no homology and high-efficiency zero background.
The technical problems to be solved by the invention are as follows:
in the prior art, because gene segments have no homology, the 6 segments can not be sequentially connected into a ring at one time, and the conventional gene synthesis method is completed by segmenting and sequentially carrying out multiple steps, so that more steps of a round of managers are required.
The purpose of the invention can be realized by the following technical scheme:
a method for high-efficiency zero-background group with no homology and multiple long fragments, comprising the following steps:
firstly, adding a B fragment Yescari at the unique position of the NotI site of the restriction endonuclease of the whole circularization plasmid, and simultaneously ensuring that the left and the right of the B fragment have just two NotI enzyme cutting sites so as to remove the B fragment by the single cutting of the subsequent NotI;
secondly, designing two Primer1/2 primers among A, B fragments without homology, designing two Primer3/4 primers among B, C fragments without homology, using the primers as bridges connected between the two fragments, and designing the rest fragments in sequence;
thirdly, 7 ABCDEFGs are obtained by PCR amplification or plasmid enzyme digestion of all the fragments, 7 primer pairs 1-14 are obtained by the same primer pair at the joint of each fragment, the fragments and the primer pairs are transferred into yeast competent cells together, and the yeast competent cells are subjected to shake culture at 30 ℃ for two days by using Sc-Trp tryptophan-deficient liquid culture medium;
fourthly, extracting and culturing yeast plasmids for two days;
fifthly, transforming the extracted yeast plasmid into escherichia coli for amplification;
sixthly, PCR bacterial examination, extraction of escherichia coli plasmid enzyme digestion verification and sequencing verification;
seventhly, singly cutting correct escherichia coli plasmids by NotI, recovering by gel electrophoresis, removing B fragments, introducing the escherichia coli plasmids into the escherichia coli for culture and screening at 37 ℃ after self-ligation treatment by T4 DNA ligase to obtain the plasmids with redundant B fragments removed, finally extracting the plasmids in the escherichia coli, and verifying the correctness of the plasmids by sequencing and enzyme digestion.
Further, the specific operation steps for extracting the yeast plasmid are as follows:
step S1, taking 1-5mL of yeast culture, centrifuging for 1-3min at the rotating speed of 12000rpm, and sucking the supernatant;
step S2, breaking yeast cell wall: adding 470 μ L sorbitol Buffer into yeast, suspending thallus sufficiently, adding 25 μ L yeast broken wall mold and 5 μ L beta-mercaptoethanol, mixing, processing at 30 deg.C for 1-2 hr while inverting centrifuge tube for 3-5 times, centrifuging at 12000r/min for 1-3min, discarding supernatant, collecting precipitate, adding 250 μ L LYP1 into the precipitate, suspending the precipitate sufficiently, and sucking the supernatant;
step S3, adding 350 mu L of YP3 into a centrifuge tube, immediately and gently turning the centrifuge tube up and down for 6 to 8 times, uniformly mixing, centrifuging the mixture for 10min at a rotating speed of 12000r/min, and carefully transferring the supernatant into another clean centrifuge tube by using a pipette;
and S4, adding the supernatant obtained in the step S3 into an adsorption column, standing at room temperature for 2min, centrifuging at 12000r/min for 1min, pouring off waste liquid in the collection tube, putting the adsorption column back into the collection tube again, adding 600 mu L of rinsing liquid into the adsorption column, centrifuging at 12000rpm for 1min, removing the waste liquid, putting the adsorption column into the collection tube, centrifuging at 12000r/min for 2min, putting the adsorption column in a room temperature or 50 ℃ incubator for 3-5min after opening the adsorption column, putting the adsorption column into a clean centrifugal tube, suspending and dropwise adding 5-200 mu L of eluent preheated by a 65 ℃ water bath into the center of the adsorption membrane, standing at room temperature for 2min, and centrifuging at 12000r/min for 1min to obtain the yeast plasmid.
The invention has the beneficial effects that:
the invention provides a method for efficiently cloning zero background group without homology, multiple long fragments, wherein zero background cloning is completed through yeast assembly and escherichia coli screening, no empty vector is produced, and a large amount of time is saved compared with the traditional cloning mode.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the structure of the primer1/2 design of the adaptor primers of fragment A and B of the present invention;
FIG. 2 shows the design of fragment B and C adaptor primer3/4 according to the invention;
FIG. 3 is a diagram of the fragments and primers of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Referring to FIGS. 1-3, a method for high efficiency zero background panel with multiple long fragments without homology comprises the following steps:
firstly, adding a B fragment Yescari at the unique position of the NotI site of the restriction endonuclease of the whole circularization plasmid, and simultaneously ensuring that the left and the right of the B fragment have just two NotI enzyme cutting sites so as to remove the B fragment by the single cutting of the subsequent NotI;
secondly, designing two Primer1/2 primers among A, B fragments without homology, designing two Primer3/4 primers among B, C fragments without homology, using the primers as bridges connected between the two fragments, and designing the rest fragments in sequence;
thirdly, 7 ABCDEFGs are obtained by PCR amplification or plasmid enzyme digestion of all the fragments, 7 primer pairs 1-14 are obtained by the same primer pair at the joint of each fragment, the fragments and the primer pairs are transferred into yeast competent cells together, and the yeast competent cells are subjected to shake culture at 30 ℃ for two days by using Sc-Trp tryptophan-deficient liquid culture medium;
fourthly, extracting and culturing yeast plasmids for two days;
fifthly, transforming the extracted yeast plasmid into escherichia coli for amplification;
sixthly, PCR bacterial examination, extraction of escherichia coli plasmid enzyme digestion verification and sequencing verification;
seventhly, singly cutting correct escherichia coli plasmids by NotI, recovering by gel electrophoresis, removing B fragments, introducing the escherichia coli plasmids into the escherichia coli for culture and screening at 37 ℃ after self-ligation treatment by T4 DNA ligase to obtain the plasmids with redundant B fragments removed, finally extracting the plasmids in the escherichia coli, and verifying the correctness of the plasmids by sequencing and enzyme digestion.
Wherein the specific operation steps for extracting the yeast plasmid are as follows:
s1, taking 1mL of yeast culture, centrifuging for 1min at the rotating speed of 12000rpm, and sucking the supernatant;
step S2, breaking yeast cell wall: adding 470 mu L sorbitol Buffer into yeast, suspending thallus fully, adding 25 mu L yeast broken wall mold and 5 mu L beta-mercaptoethanol, mixing, processing at 30 deg.C for 1h, inverting centrifuge tube for 3 times, centrifuging at 12000r/min for 1min, discarding supernatant, collecting precipitate, adding 250 mu LYP1 into the precipitate, suspending the precipitate fully, and sucking the supernatant;
step S3, adding 350 mu L of YP3 into a centrifuge tube, immediately turning the centrifuge tube up and down for 6 times gently, mixing the centrifuge tube uniformly, centrifuging the centrifuge tube for 10min at a rotating speed of 12000r/min, and carefully transferring the supernatant into another clean centrifuge tube by using a pipette;
and S4, adding the supernatant obtained in the step S3 into an adsorption column, standing at room temperature for 2min, centrifuging at the rotating speed of 12000r/min for 1min, pouring the waste liquid in the collecting pipe, putting the adsorption column back into the collecting pipe again, adding 600 mu L of rinsing liquid into the adsorption column, centrifuging at 12000rpm for 1min, removing the waste liquid, putting the adsorption column into the collecting pipe, centrifuging at the rotating speed of 12000r/min for 2min, putting the adsorption column in a room temperature or 50 ℃ incubator for 3min after opening the adsorption column, putting the adsorption column into a clean centrifugal pipe, hanging and dripping 5 mu L of eluent preheated by a 65 ℃ water bath into the center of an adsorption membrane, standing at room temperature for 2min, and centrifuging at 12000r/min for 1min to obtain the yeast plasmid.
Example 2
A method for high-efficiency zero-background group with no homology and multiple long fragments, comprising the following steps:
firstly, adding a B fragment Yescari at the unique position of the NotI site of the restriction endonuclease of the whole circularization plasmid, and simultaneously ensuring that the left and the right of the B fragment have just two NotI enzyme cutting sites so as to remove the B fragment by the single cutting of the subsequent NotI;
secondly, designing two Primer1/2 primers among A, B fragments without homology, designing two Primer3/4 primers among B, C fragments without homology, using the primers as bridges connected between the two fragments, and designing the rest fragments in sequence;
thirdly, 7 ABCDEFGs are obtained by PCR amplification or plasmid enzyme digestion of all the fragments, 7 primer pairs 1-14 are obtained by the same primer pair at the joint of each fragment, the fragments and the primer pairs are transferred into yeast competent cells together, and the yeast competent cells are subjected to shake culture at 30 ℃ for two days by using Sc-Trp tryptophan-deficient liquid culture medium;
fourthly, extracting and culturing yeast plasmids for two days;
fifthly, transforming the extracted yeast plasmid into escherichia coli for amplification;
sixthly, PCR bacterial examination, extraction of escherichia coli plasmid enzyme digestion verification and sequencing verification;
seventhly, singly cutting correct escherichia coli plasmids by NotI, recovering by gel electrophoresis, removing B fragments, introducing the escherichia coli plasmids into the escherichia coli for culture and screening at 37 ℃ after self-ligation treatment by T4 DNA ligase to obtain the plasmids with redundant B fragments removed, finally extracting the plasmids in the escherichia coli, and verifying the correctness of the plasmids by sequencing and enzyme digestion.
Wherein the specific operation steps for extracting the yeast plasmid are as follows:
s1, taking 3mL of yeast culture, centrifuging for 2min at the rotating speed of 12000rpm, and sucking a supernatant;
step S2, breaking yeast cell wall: adding 470 mu L sorbitol Buffer into yeast, suspending thallus fully, adding 25 mu L yeast broken wall mold and 5 mu L beta-mercaptoethanol, mixing, processing at 30 deg.C for 1.5h, reversing centrifuge tube 4 times, centrifuging at 12000r/min for 2min, discarding supernatant, collecting precipitate, adding 250 mu LYP1 into the precipitate, suspending the precipitate fully, and sucking the supernatant;
step S3, adding 350 mu L of YP3 into a centrifuge tube, immediately and gently turning the centrifuge tube up and down for 7 times, uniformly mixing, centrifuging the centrifuge tube at a rotating speed of 12000r/min for 10min, and carefully transferring the supernatant into another clean centrifuge tube by using a pipette;
and S4, adding the supernatant obtained in the step S3 into an adsorption column, standing at room temperature for 2min, centrifuging at the rotating speed of 12000r/min for 1min, pouring the waste liquid in the collecting tube, putting the adsorption column back into the collecting tube again, adding 600 mu L of rinsing liquid into the adsorption column, centrifuging at 12000rpm for 1min, removing the waste liquid, putting the adsorption column into the collecting tube, centrifuging at the rotating speed of 12000r/min for 2min, putting the adsorption column in a room temperature or 50 ℃ incubator for 4min after opening the adsorption column, putting the adsorption column into a clean centrifugal tube, hanging and dropwise adding 100 mu L of eluent preheated by a 65 ℃ water bath into the center of the adsorption membrane, standing at room temperature for 2min, and centrifuging at 12000r/min for 1min to obtain the yeast plasmid.
Example 3
A method for high-efficiency zero-background group with no homology and multiple long fragments, comprising the following steps:
firstly, adding a B fragment Yescari at the unique position of the NotI site of the restriction endonuclease of the whole circularization plasmid, and simultaneously ensuring that the left and the right of the B fragment have just two NotI enzyme cutting sites so as to remove the B fragment by the single cutting of the subsequent NotI;
secondly, designing two Primer1/2 primers among A, B fragments without homology, designing two Primer3/4 primers among B, C fragments without homology, using the primers as bridges connected between the two fragments, and designing the rest fragments in sequence;
thirdly, 7 ABCDEFGs are obtained by PCR amplification or plasmid enzyme digestion of all the fragments, 7 primer pairs 1-14 are obtained by the same primer pair at the joint of each fragment, the fragments and the primer pairs are transferred into yeast competent cells together, and the yeast competent cells are subjected to shake culture at 30 ℃ for two days by using Sc-Trp tryptophan-deficient liquid culture medium;
fourthly, extracting and culturing yeast plasmids for two days;
fifthly, transforming the extracted yeast plasmid into escherichia coli for amplification;
sixthly, PCR bacterial examination, extraction of escherichia coli plasmid enzyme digestion verification and sequencing verification;
seventhly, singly cutting correct escherichia coli plasmids by NotI, recovering by gel electrophoresis, removing B fragments, introducing the escherichia coli plasmids into the escherichia coli for culture and screening at 37 ℃ after self-ligation treatment by T4 DNA ligase to obtain the plasmids with redundant B fragments removed, finally extracting the plasmids in the escherichia coli, and verifying the correctness of the plasmids by sequencing and enzyme digestion.
Wherein the specific operation steps for extracting the yeast plasmid are as follows:
s1, taking 5mL of yeast culture, centrifuging for 3min at the rotating speed of 12000rpm, and sucking a supernatant;
step S2, breaking yeast cell wall: adding 470 mu L sorbitol Buffer into yeast, suspending thallus fully, adding 25 mu L yeast broken wall mold and 5 mu L beta-mercaptoethanol, mixing, processing at 30 deg.C for 2h, reversing centrifuge tube for 5 times, centrifuging at 12000r/min for 3min, discarding supernatant, collecting precipitate, adding 250 mu LYP1 into the precipitate, suspending the precipitate fully, and sucking the supernatant;
step S3, adding 350 mu L of YP3 into a centrifuge tube, immediately and gently turning the centrifuge tube up and down for 8 times, uniformly mixing, centrifuging the centrifuge tube at a rotating speed of 12000r/min for 10min, and carefully transferring the supernatant into another clean centrifuge tube by using a pipette;
and S4, adding the supernatant obtained in the step S3 into an adsorption column, standing at room temperature for 2min, centrifuging at the rotating speed of 12000r/min for 1min, pouring the waste liquid in the collecting tube, putting the adsorption column back into the collecting tube again, adding 600 mu L of rinsing liquid into the adsorption column, centrifuging at 12000rpm for 1min, removing the waste liquid, putting the adsorption column into the collecting tube, centrifuging at the rotating speed of 12000r/min for 2min, putting the adsorption column in a room temperature or 50 ℃ incubator for 5min after opening the adsorption column, putting the adsorption column into a clean centrifugal tube, hanging 200 mu L of eluent preheated by a 65 ℃ water bath in the middle of the adsorption membrane, standing at room temperature for 2min, and centrifuging at 12000r/min for 1min to obtain the yeast plasmid.
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.
Claims (2)
1. The method for the efficient zero background group without homology and multiple long fragments is characterized by comprising the following steps of:
firstly, adding a B fragment Yescari at the unique position of the NotI site of the restriction endonuclease of the whole circularization plasmid, and simultaneously ensuring that the left and the right of the B fragment have just two NotI enzyme cutting sites so as to remove the B fragment by the single cutting of the subsequent NotI;
secondly, designing two Primer1/2 primers among A, B fragments without homology, designing two Primer3/4 primers among B, C fragments without homology, using the primers as bridges connected between the two fragments, and designing the rest fragments in sequence;
thirdly, transferring 7 ABCDEFGs of all the fragments and 7 primer pairs 1-14 of a primer pair at the joint of each fragment into yeast competent cells together, and performing shake culture at 30 ℃ for two days by using Sc-Trp tryptophan-deficient liquid culture medium;
fourthly, extracting and culturing yeast plasmids for two days;
fifthly, transforming the extracted yeast plasmid into escherichia coli for amplification;
sixthly, PCR bacterial examination, extraction of escherichia coli plasmid enzyme digestion verification and sequencing verification;
seventhly, singly cutting correct escherichia coli plasmids by NotI, recovering by gel electrophoresis, removing B fragments, introducing the escherichia coli plasmids into the escherichia coli for culture and screening at 37 ℃ after self-ligation treatment by T4 DNA ligase to obtain the plasmids with redundant B fragments removed, finally extracting the plasmids in the escherichia coli, and verifying the correctness of the plasmids by sequencing and enzyme digestion.
2. The method of claim 1, wherein the specific steps for extracting yeast plasmids are as follows:
step S1, taking 1-5mL of yeast culture, centrifuging for 1-3min at the rotating speed of 12000rpm, and sucking the supernatant;
step S2, breaking yeast cell wall: adding 470 μ L sorbitol Buffer into yeast, suspending thallus sufficiently, adding 25 μ L yeast broken wall mold and 5 μ L beta-mercaptoethanol, mixing, processing at 30 deg.C for 1-2 hr while inverting centrifuge tube for 3-5 times, centrifuging at 12000r/min for 1-3min, discarding supernatant, collecting precipitate, adding 250 μ L LYP1 into the precipitate, suspending the precipitate sufficiently, and sucking the supernatant;
step S3, adding 350 mu L of YP3 into a centrifuge tube, immediately and gently turning the centrifuge tube up and down for 6 to 8 times, uniformly mixing, centrifuging the mixture for 10min at a rotating speed of 12000r/min, and carefully transferring the supernatant into another clean centrifuge tube by using a pipette;
and S4, adding the supernatant obtained in the step S3 into an adsorption column, standing at room temperature for 2min, centrifuging at 12000r/min for 1min, pouring off waste liquid in the collection tube, putting the adsorption column back into the collection tube again, adding 600 mu L of rinsing liquid into the adsorption column, centrifuging at 12000rpm for 1min, removing the waste liquid, putting the adsorption column into the collection tube, centrifuging at 12000r/min for 2min, putting the adsorption column in a room temperature or 50 ℃ incubator for 3-5min after opening the adsorption column, putting the adsorption column into a clean centrifugal tube, suspending and dropwise adding 5-200 mu L of eluent preheated by a 65 ℃ water bath into the center of the adsorption membrane, standing at room temperature for 2min, and centrifuging at 12000r/min for 1min to obtain the yeast plasmid.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN104313044A (en) * | 2014-09-30 | 2015-01-28 | 天根生化科技（北京）有限公司 | Zero-background cloning vector as well as preparation method and application thereof |
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