CN104404077A - Method for simultaneously cloning multiple exogenous genes to microbial genome - Google Patents
Method for simultaneously cloning multiple exogenous genes to microbial genome Download PDFInfo
- Publication number
- CN104404077A CN104404077A CN201410621447.0A CN201410621447A CN104404077A CN 104404077 A CN104404077 A CN 104404077A CN 201410621447 A CN201410621447 A CN 201410621447A CN 104404077 A CN104404077 A CN 104404077A
- Authority
- CN
- China
- Prior art keywords
- seq
- expression vector
- way
- sequence table
- bidirectional
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for simultaneously cloning multiple exogenous genes to a microbial genome. The method comprises steps of introducing a plurality of exogenous genes to a plurality of bidirectional gene expression carriers, and then simultaneously introducing the plurality of bidirectional gene expression carriers into a host microbe. Each bidirectional gene expression carrier comprises a bidirectional terminator and a bidirectional promoter. Except for the first bidirectional gene expression carrier and the last bidirectional gene expression carrier, the 3' ends of the bidirectional terminators of other bidirectional gene expression carriers and the 5' ends of the bidirectional terminators of the next bidirectional gene expression carriers have the same homologous arm. The 5' end of the bidirectional terminator of the first bidirectional gene expression carrier and the 3' end of the bidirectional terminator of the last bidirectional gene expression carrier can both carry out homologous recombination with the genomes of a host microbe. Through the provided method, established is a novel technology that can organically integrate the whole gene expression process such as expression design, clone design, host cell modification, and the like.
Description
Technical field
The invention belongs to genetically engineered field, relate to a kind of method multiple foreign gene being cloned into simultaneously microbial genome.
Background technology
The transformation of microbial DNA, comprising knocking out of target gene is the core content in genetically engineered field with process LAN.The expression of one or two gene in target microorganism is not difficult to realize by prior art, but, the expression of polygene (two or more) remains in many difficult problems according to prior art, and as cloned, the cycle is long, workload is large, success ratio is low, genetic expression transformation is low.Especially Eukaryotic genetic expression, general each gene needs independent promotor and terminator, a genetic expression is made just to need to carry out 3 time clonings, if carry out multi-gene expression, as 6 genetic expressions, just need to carry out 18 gene clones, this situation for use traditional method be difficult to realize and very time-consuming.
At present, some emerging clone technologies, although can improve the efficiency of clone to a certain extent as Gateway recombinant technology, Gibson package technique and Golden gate technology etc., also there is certain short slab or restriction separately in it in application.What is more important, these novel methods, just for this sport technique segment of gene clone, do not provide any help in sequences Design, design of expression and transformed host cell etc.Therefore, need a kind of new technology by whole genetic expression process, comprise the process such as design of expression, clone design, transform host cell and organically combine, realize polygenic quick, high expression, Getting Started Steps threshold can be reduced simultaneously, reduce the training and learning time, reduce running cost.
At present many relate to genetic expression patented technology or tool carrier (plasmid) the quick expression of one to two genes can be realized.But the carrier in these patents is generally contriver build with fixing promotor or other transcriptional elements, the expression of target gene can only be carried out with fixing intensity.And in current genetic engineering modified work, often need polygenic cooperation, namely need each Gene expression intensities to create a difference according to planner's wish, this is that existing tool carrier or technology are difficult to realize, and this technology will solve this difficult problem.
In addition, apigenin have antitumor, improve cardiac and cerebral blood-supply, anti-inflammatory, hypotensive, anxiety, anti-bacteria and anti-virus and the physiological function such as anti-oxidant.Yeast saccharomyces cerevisiae self can not produce apigenin, can by phenylalanine synthetic celery element after transforming apigenin synthetic gene (PAL, C4H, 4CL, CHS, CHI and FSII).Therefore, a kind of method that apigenin synthetic gene can be imported fast yeast saccharomyces cerevisiae is needed.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, provides a kind of and multiple foreign gene is cloned into microbial genome simultaneously, realizes the method for multi-gene expression and microbial gene transformation.
The invention provides a kind of method multiple foreign gene being cloned into simultaneously microbial genome, multiple foreign gene is comprised the steps: first to import multiple two-way expression vector, again multiple two-way expression vector is imported host microorganism simultaneously, obtain the recombinant microorganism of expressing multiple foreign gene;
Described each two-way expression vector includes a two-way terminator and a bidirectional promoter; Each two-way expression vector imports two foreign genes;
Except first two-way expression vector and last two-way expression vector, 3 ' end of other each two-way expression vectors and 5 ' end of the two-way expression vector of the next one have identical homology arm, 5 ' end of described first two-way expression vector has homology bracelet, 3 ' end of last two-way expression vector described has homology arm second, described homology bracelet and described homology arm second all can with the genome generation homologous recombination of host microorganism.
Further, described host microorganism is yeast; Be preferably yeast saccharomyces cerevisiae, be more preferably yeast saccharomyces cerevisiae W303.
Described multiple foreign gene is 4CL, CHS, CHI, FSII, PAL, C4H gene, and its gene order is successively as shown in sequence table SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11.
Described two-way expression vector has three, first two-way expression vector comprises the two-way terminator T1 (TPI1-PGIt) as shown in SEQ ID NO:1 in sequence table, and the bidirectional promoter P1 (TDH3-ADH1) in sequence table shown in SEQ ID NO:4; Second two-way expression vector comprises the two-way terminator T2 (ADH1t-CYC1t) as shown in SEQ ID NO:2 in sequence table, and the bidirectional promoter P2 (PGK1-TEF2) in sequence table shown in SEQ ID NO:5; 3rd two-way expression vector comprises the two-way terminator T3 (tFBA1-tPDC1) as shown in SEQ ID NO:3 in sequence table, and the bidirectional promoter P1 (TDH3-ADH1) in sequence table shown in SEQ ID NO:4.
Wherein, the two-way terminator T1 (TPI1-PGIt) of the first two-way expression vector, as shown in sequence table SEQ ID NO:1, from the Nucleotide of 5 ' end the 1131st to 1180, with the two-way terminator T2 (ADH1t-CYC1t) of the second two-way expression vector, as shown in sequence table SEQ ID NO:2, be homology arm L2 from the Nucleotide of 5 ' end the 1st to 150.
The two-way terminator T2 (ADH1t-CYC1t) of the second two-way expression vector, as shown in sequence table SEQ ID NO:2, from the Nucleotide of 5 ' end the 627th to 776, with the two-way terminator T3 (tFBA1-tPDC1) of the 3rd two-way expression vector, as shown in sequence table SEQ IDNO:4, be homology arm L3 from the Nucleotide of 5 ' end the 1st to 150.
Described first two-way expression vector also comprises described 4CL, CHS gene; Described second two-way expression vector also comprises described CHI, FSII gene; Described 3rd two-way expression vector also comprises described PAL, C4H gene.
In aforesaid method, the preparation of the first two-way expression vector, the second two-way expression vector, the 3rd two-way expression vector comprises the following steps:
(1), SEQ ID NO:1, SEQ ID NO:2, the two-way terminator T1 (TPI1-PGIt) shown in SEQ ID NO:3, two-way terminator T2 (ADH1t-CYC1t), two-way terminator T3 (tFBA1-tPDC1) in composition sequence table, respectively by 1180,776,963 based compositions; Bidirectional promoter P2 (PGK1-TEF2) shown in SEQ ID NO:5 in bidirectional promoter P1 (TDH3-ADH1) in composition sequence table shown in SEQ ID NO:4, sequence table, respectively by 1418,1312 based compositions.
(2), by two-way terminator T1 (TPI1-PGIt), two-way terminator T2 (ADH1t-CYC1t), two-way terminator T3 (tFBA1-tPDC1) be connected on pUC57-Kan plasmid, obtain recombinant expression vector p-T1 (TPI1-PGIt), p-T2 (ADH1t-CYC1t), p-T3 (tFBA1-tPDC1); Bidirectional promoter P1 (TDH3-ADH1), bidirectional promoter P2 (PGK1-TEF2) are connected on PMD18-T plasmid and obtain recombinant expression vector PMD-P1 (TDH3-ADH1), PMD-P2 (PGK1-TEF2).
(3), SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,4CL, CHS, CHI, FSII, PAL, C4H gene shown in SEQ ID NO:10, SEQID NO:11 in composition sequence table, said gene all comes from feverfew Erigeron breviscapus (Vant.) Hand.-Mazz. Erigeronbreviscapus (Vant.) Hand-Mazz, it has the gene order of yeast saccharomyces cerevisiae preference codon after optimizing, respectively by 1624,1199,718,1562,2136,2136 based compositions.
(4), with the recombinant expression vector p-T1 (TPI1-PGIt) of step (2) gained, p-T3 (tFBA1-tPDC1) and host microorganism genes of brewing yeast group for template, build homology bracelet L1, homology arm second L4, integration site Site1, Site2, marker gene HIS fragment and overlapping fragments Site1-His-L1 and Site2-L4.
(5), the first two-way expression vector p-T1 (4CL-CHS), the second two-way expression vector p-T2 (CHI-FSII) and the 3rd two-way expression vector p-T3 (PAL-C4H) is built;
Two-way terminator T1 (TPI1-PGIt), bidirectional promoter P1 (TDH3-ADH1) and 4CL, CHS gene are connected and composed the first two-way expression vector p-T1 (4CL-CHS);
Two-way terminator T2 (ADH1t-CYC1t), bidirectional promoter P2 (PGK1-TEF2) and CHI, FSII gene are connected and composed the second two-way expression vector p-T2 (CHI-FSII);
Two-way terminator T3 (tFBA1-tPDC1), bidirectional promoter P1 (TDH3-ADH1) and PAL, C4H gene are connected and composed the 3rd two-way expression vector p-T3 (PAL-C4H).
Further, described three two-way expression vectors import host microorganism yeast saccharomyces cerevisiae w303 simultaneously, obtain recombinant microorganism called after recombinant Saccharomyces cerevisiae W303-1.
Described recombinant Saccharomyces cerevisiae W303-1 is classified as the recombinant Saccharomyces cerevisiae of the DNA fragmentation shown in SEQ ID NO:6-11 in sequence table for expressing nucleotides sequence.
The invention provides a kind of method multiple foreign gene one step be incorporated in microbe on genome, by the principle of homologous recombination, utilize two-way expression vector, required multiple expression vectors are imported in host cell simultaneously, establish a kind of by whole genetic expression process, comprise the new technology that the process such as design of expression, clone design, transform host cell organically combines, achieve polygenic quick, high expression, avoid the trouble that repeatedly conversion and vector construction bring.
Accompanying drawing explanation
Fig. 1 is pUC57-Kan plasmid.
Fig. 2 is recombinant expression vector p-T1 (TPI1-PGIt) plasmid.
Fig. 3 is recombinant expression vector p-T2 (ADH1t-CYC1t) plasmid.
Fig. 4 is recombinant expression vector p-T3 (tFBA1-tPDC1) plasmid.
Fig. 5 is PMD18-T plasmid.
Fig. 6 is recombinant expression vector PMD-P1 (TDH3-ADH1) plasmid.
Fig. 7 is recombinant expression vector PMD-P2 (PGK1-TEF2) plasmid.
Fig. 8 is homology arm L1, L4 in embodiment 4, integration site Site1, Site2, the result of the agarose gel electrophoresis purifying after the amplification of marker gene HIS fragment PCR.
Fig. 9 is the agarose gel electrophoresis result in embodiment 5 after overlapping fragments Site1-His-L1 and Site2-L4PCR amplification.
Figure 10 introduces the agarose gel electrophoresis result after restriction enzyme site pcr amplification after the amplification of PAL, C4H gene PCR in embodiment 6.
Figure 11 is the agarose gel electrophoresis result introducing restriction enzyme site pcr amplification 4CL, CHS, CHI, FSII gene in embodiment 6.
Figure 12 is the agarose gel electrophoresis result of two-way terminator gene T1 (TPI1-PGIt), T2 (ADH1t-CYC1t), T3 (tFBA1-tPDC1) after introducing restriction enzyme site pcr amplification in embodiment 6.
Figure 13 is the agarose gel electrophoresis result introducing bidirectional promoter gene P1 (TDH3-ADH1) after restriction enzyme site pcr amplification, P2 (PGK1-TEF2) in embodiment 6.
Figure 14 is the agarose gel electrophoresis result of colibacillus PCR amplification 4CL, CHS gene in embodiment 6.
Figure 15 is the agarose gel electrophoresis result of colibacillus PCR amplification CHI, FSII gene in embodiment 6.
Figure 16 is the agarose gel electrophoresis result of colibacillus PCR amplification PAL, C4H gene in embodiment 6.
Figure 17 is the agarose gel electrophoresis result of p-T1 (4CL-CHS), p-T2 (CHI-FSII), p-T3 (PAL-C4H) after enlarged culturing in embodiment 6.
Figure 18 is the agarose gel electrophoresis result of T1 (4CL-CHS), T2 (CHI-FSII) after pcr amplification in embodiment 7.
Figure 19 is the agarose gel electrophoresis result of T3 (PAL-C4H) after pcr amplification in embodiment 7.
Figure 20 is agarose gel electrophoresis result after recombinant Saccharomyces cerevisiae W303-1 genomic PCR amplification in embodiment 9.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
The synthesis of the two-way terminator of embodiment 1, bidirectional promoter
(1) synthesis of two-way terminator
Two-way terminator T1 (TPI1-PGIt) in composition sequence table shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, two-way terminator T2 (ADH1t-CYC1t), two-way terminator T3 (tFBA1-tPDC1), respectively by 1180,776,963 based compositions.Wherein, the two-way terminator T1 (TPI1-PGIt) of the first two-way expression vector, as shown in sequence table SEQ ID NO:1, from the Nucleotide of 5 ' end the 1031st to 1180, with the two-way terminator T2 (ADH1t-CYC1t) of the second two-way expression vector, as shown in sequence table SEQ ID NO:2, be homology arm L2 from the Nucleotide of 5 ' end the 1st to 150.The two-way terminator T2 (ADH1t-CYC1t) of the second two-way expression vector, as shown in sequence table SEQ ID NO:2, from the Nucleotide of 5 ' end the 627th to 776, with the two-way terminator T3 (tFBA1-tPDC1) of the 3rd two-way expression vector, as shown in sequence table SEQ ID NO:4, be equal homology arm L3 from the Nucleotide of 5 ' end the 1st to 150.
(2) synthesis of bidirectional promoter
Bidirectional promoter P2 (PGK1-TEF2) shown in SEQ ID NO:5 in bidirectional promoter P1 (TDH3-ADH1) in composition sequence table shown in SEQ ID NO:4, sequence table, respectively by 1418,1312 based compositions.
Embodiment 2 builds p-T1 (TPI1-PGIt) plasmid, p-T2 (ADH1t-CYC1t) plasmid, p-T3 (tFBA1-tPDC1) plasmid, PMD-P1 (TDH3-ADH1) plasmid, PMD-P2 (PGK1-TEF2) plasmid
Two-way terminator T1 (TPI1-PGIt), two-way terminator T2 (ADH1t-CYC1t), two-way terminator T3 (tFBA1-tPDC1) are connected on pUC57-Kan plasmid (as shown in Figure 1), obtain recombinant expression vector p-T1 (TPI1-PGIt) (as shown in Figure 2), p-T2 (ADH1t-CYC1t) (as shown in Figure 3), p-T3 (tFBA1-tPDC1) (as shown in Figure 4).Bidirectional promoter P1 (TDH3-ADH1), bidirectional promoter P2 (PGK1-TEF2) are connected on PMD18-T plasmid (as shown in Figure 5) and obtain recombinant expression vector PMD-P1 (TDH3-ADH1) (as shown in Figure 6), PMD-P2 (PGK1-TEF2) (as shown in Figure 7).
The synthesis of embodiment 3 goal gene
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,4CL, CHS, CHI, FSII, PAL, C4H gene shown in SEQ ID NO:11 in composition sequence table, said gene all comes from feverfew Erigeron breviscapus (Vant.) Hand.-Mazz. Erigeronbreviscapus (Vant.) Hand-Mazz, it has the gene order of yeast saccharomyces cerevisiae preference codon after optimizing, respectively by 1624,1199,718,1562,2136,2136 based compositions.
Embodiment 4 homology arm L1, L4, integration site Site1, Site2, the structure of marker gene HIS fragment
(1) design of primers:
L1-Fo:5’-TCCCTCCACCAAAGGTGTTCTTATGTAGCCCCGGTCCGTTTGTTCTAT-3’
L1-Ro:5’-CTCGAGAGGCAATTTTAGAGGGGACTTC-3’
L4-Fo:5’-CCAGACGATACAGAGGCTAAGA-3’；
L4-Ro:5’-CCACTTTTGTTGGGGACGATTAGGTTCCAACTGCTCTTACTGT-3’
Site1-Fo:5’-GTCGGTCGACTGTGCACAAAGGCCATAATAT-3’
Site1-Ro:5’-TCGGAAGAGGTTTTGTCATCCGAAGGCATGAGTTATGGTTGCACA-3’
Site2-Fo:5’-TACAGTAAGAGCAGTTGGAACCTAATCGTCCCCAACAAAAGTGGGCT-3’
Site2-Ro:5’-AAAGCTGGCTCCCCTTAGACAAATACGCTA-3’
HIS-Fo:5’-AACCATAACTCATGCCTTCGGATGACAAAACCTCTTCCGATAAAAACA-3’
HIS-Ro:5’-TAGAACAAACGGACCGGGGCTACATAAGAACACCTTTGGTGGAGGGA-3’
(2) PCR reaction system:
| Upstream primer | Downstream primer | Template | The extension time |
| Site1-Fo | Site1-Ro | Yeast saccharomyces cerevisiae W303 genome | 30 seconds |
| Site2-Fo | Site2-Ro | Yeast saccharomyces cerevisiae W303 genome | 30 seconds |
| HIS-Fo | HIS-Ro | P-YES2 | 1 point 15 seconds |
| L1-Fo | L1-Ro | p-T1(TPI1-PGIt) | 20 seconds |
| L4-Fo | L4-Ro | p-T3(tFBA1-tPDC1) | 20 seconds |
(3) pcr amplification condition: 94 DEG C of sex change 5 minutes, afterwards " 94 degree of sex change, 30 seconds, 58 DEG C annealing 30 seconds, 72 DEG C extend 1 point 15 seconds " 34 circulations, 72 DEG C extend 10min, 4 DEG C of insulations.
(4) agarose gel electrophoresis purifying, glue figure result as shown in Figure 8.
The structure of embodiment 5 overlapping fragments Site1-His-L1 and Site2-L4
(1) structure of overlapping fragments site1-His-L1
A () PCR system: site1 1ul, His 1ul, L1 1ul, site1-F 2ul, L1-R 2ul, TOP Taq 0.5ul, 10Xbuffer5ul, dNTPs 4ul sterilized water mend final volume 50ul.B () PCR response procedures: 94 DEG C of sex change 5 minutes, afterwards " 94 degree of sex change, 30 seconds, 58 DEG C annealing 30 seconds, 72 DEG C extend 1 point 30 seconds " 34 circulations, 72 DEG C extend 10min, 4 DEG C of insulations.C () agarose gel electrophoresis purifying, glue figure result as shown in Figure 9.
(2) structure of overlapping fragments Site2-L4
A () PCR system: site2 1ul, L4 1ul, site1-F 2ul, L1-R 2ul, TOP Taq 0.5ul, 10Xbuffer 5ul, dNTPs 4ul sterilized water mend final volume 50ul.B () PCR response procedures: 94 DEG C of sex change 5 minutes, afterwards " 94 degree of sex change, 30 seconds, 58 DEG C annealing 30 seconds, 72 DEG C extend 50 seconds " 34 circulations, 72 DEG C extend 10min, 4 DEG C of insulations.C () agarose gel electrophoresis purifying, glue figure result as shown in Figure 9.
The structure of the two-way expression vector p-T1 (4CL-CHS) of embodiment 6, p-T2 (CHI-FSII), p-T3 (PAL-C4H)
(1) on goal gene 4CL, CHS, CHI, FSII, PAL, C4H, BsaI restriction enzyme site is introduced respectively:
(a) design of primers:
CHS-F：5’-ACCAGGTCTCAGAATGGCTTCTTCTATCGACATCGCTGC-3’
CHS-R：5’-ACCAGGTCTCACGATTTAAGTAGCGATAGCAGTAGTAGTTGGCAAAGAGT-3’
4CL-F：5’-ACCAGGTCTCAAGGTTTAAGATGGAACACCAGCAGCCA-3’
4CL-R：5’-ACCAGGTCTCAGATGGACTCTCAAAAGGAAATCATCTTCAGATCT-3’
CHI-F：5’-ACCAGGTCTCAAGGTTTACAAACCGTACTTAGAAACGTCAGC-3’
CHI-R：5’-ACCAGGTCTCAGATGGCTGCTACTACTACTCCATTGAC-3’
FSII-F：5’-ACCAGGTCTCAGAATGAACATGTTGCAAGTTTTCCAATC-3’
FSII-R：5’-ACCAGGTCTCACGATTTAAGTAGAACCCAAGATTTGAGAACAG-3’
PAL-F：5’-CCGGTCTCAGAATATGGAAAACGGTCACGCTAACGGTG-3’
PAL-R：5’-CCGGTCTCAAGGTTTAACAGATTGGCAATGGAGCACCGTTCCAAC-3’
C4H-F：5’-CCGGTCTCAGATGATGGACTTGTTGTTGTTGGAAAAGA-3’
C4H-R：5’-CCGGTCTCACGATTTAGAAAGATCTTGGCTTAGCAACGATA-3’
(b) pcr amplification condition: with goal gene 4CL, CHS, CHI, FSII, PAL, C4H for template, 94 DEG C of sex change 5 minutes, " 94 degree of sex change 30 seconds, 58 DEG C annealing 30 seconds, 72 DEG C extend 1 point 15 seconds " 34 circulations afterwards, 72 DEG C extend 10min, 4 DEG C of insulations.
C () agarose gel electrophoresis purifying object product, result is as shown in Figure 10,11.
(2) respectively at two-way terminator gene T1 (TPI1-PGIt), T2 (ADH1t-CYC1t), T3 (tFBA1-tPDC1) and bidirectional promoter gene P1 (TDH3-ADH1), P2 (PGK1-TEF2) upper introducing BsaI restriction enzyme site.
(a) design of primers:
T1-F:5’-CAATCGAACAAATCGCTCTTAA-3’；
T1-R:5’-GAATTCGGTCTCAACCTGATTAAT-3’
T2-F:5’-AGCTTGGTCTCAATCGTCATGTAATT-3’
T2-R:5’-CGAATTCGGTCTCAACCTAGTTATA-3’
T3-F:5’-GGGTCTCAATCGGAATTCGC-3’
T3-R:5’-GGGTCTCAACCTAAGCTTGTTAA-3’
P1-F:5’-ACCAGGTCTCACATCTTTGTTTGTTTATT-3’；
P1-R:5’-ACCAGGTCTCAATTCTGTATATGAGATAGT-3’
P2-F:5’-ACCAGGTCTCACATCGTTTTATATTTGTT-3’
P2-R:5’-ACCAGGTCTCAATTCGGTACTAGTGTTTAG-3’
(b) pcr amplification condition: with two-way terminator gene T1 (TPI1-PGIt), T2 (ADH1t-CYC1t), T3 (tFBA1-tPDC1) and bidirectional promoter gene P1 (TDH3-ADH1), P2 (PGK1-TEF2) respectively 1ul for template, 94 DEG C of sex change 5 minutes, " 94 degree of sex change 30 seconds, 58 DEG C annealing 30 seconds, 72 DEG C extend 2 points 30 seconds " 34 circulations afterwards, 72 DEG C extend 10min, 4 DEG C of insulations.
C () agarose gel electrophoresis purifying object product, result as shown in Figure 12,13.
(3) cyclophorase cuts linked system
A () is one group with T1, P1,4CL, CHS, T2, P2, CHI, FSII are one group, and T3, P1, PAL, C4H are one group to be added according to following system proportioning.
| T1/T2/T3 (carrier framework) | (200ng) |
| P1/P2 | Amount equimolar with carrier framework |
| 4CL/CHI/PAL | Amount equimolar with carrier framework |
| CHS/FSII/C4H | Amount equimolar with carrier framework |
| 10XNEB T4buffer(NEB) | 1.5ul |
| 100XBSA*(NEB) | 0.15ul |
| BsaI(NEB) | 1ul |
| NEB T4Ligase(NEB) | 1ul |
| ddH2O | Supply 15ul |
| Total | 15ul |
(b) cycling program: 37 DEG C 3 minutes 16 DEG C 5 minutes, these two temperature do that 30 ~ 50 cyclophorases cut connection, 50 DEG C of 5 minutes whole enzymes are cut, 80 DEG C of deactivations in 5 minutes.
(4) enzyme is cut linked system transformation of E. coli DH5 α, the LB flat board (kantlex of 1% tryptone, 0.5% yeast extract, 1% sodium-chlor, 1.5% agar powder, 100ug/ml) of kalamycin resistance is adopted to carry out positive colony screening, 37 DEG C of incubated overnight.
(5) mono-clonal PCR verifies
A () picking list bacterium colony shakes training 2 hours (220rpm/min) in the LB liquid nutrient medium (kantlex of 1% tryptone, 0.5% yeast extract, 1% sodium-chlor, 100ug/ml) of 1ml kalamycin resistance, get 0.5ul bacterium liquid and be template PCR.
(b) PCR system:
| Upstream primer | Downstream primer | Template | The extension time |
| CHS-F | CHS-R | CHS | 1 point 15 seconds |
| 4CL-F | 4CL-R | 4CL | 1 point 15 seconds |
| CHI-F | CHI-R | CHI | 1 point 15 seconds |
| FSII-F | FSII-R | FSII | 1 point 15 seconds |
| PAL-F | PAL-R | PAL | 1 point 15 seconds |
| C4H-F | CH4-R | C4H | 1 point 15 seconds |
C () pcr amplification condition: polysaccharase is Easy Taq polysaccharase, 94 DEG C of sex change 5 minutes, afterwards " 94 degree of sex change, 30 seconds, 58 DEG C annealing 30 seconds, 72 DEG C extend 1 point 15 seconds " 34 circulations, 72 DEG C extend 10min, 4 DEG C of insulations.
D () PCR terminates agarose gel electrophoresis checking, as shown in Figure 14,15,16.
(6) plasmid enzyme restriction checking is extracted
(a) enlarged culturing: bacterium liquid 200ul corresponding for correct band is added in the LB liquid nutrient medium of 5ml kalamycin resistance and shakes training 12 ~ 16 hours (220rpm/min), enlarged culturing.
B () extracts plasmid with plasmid extraction kit to expanding the bacterium liquid shaking training acquisition.
(c) digestion verification
Enzyme cuts system: plasmid 3ul, cutsmart (enzyme cuts buffer) 2ul, BsmBI 1ul, and sterilized water supplies 20ul, and 37 DEG C of enzymes cut 2 hours.Agarose gel electrophoresis is verified, result as shown in figure 17.
The amplification of the two-way expression vector p-T1 (4CL-CHS) of embodiment 7, p-T2 (CHI-FSII), p-T3 (PAL-C4H)
(1) with the correct plasmid of digestion verification for template designs suitable primer at homology arm L1-L4 two ends
L1-F:5’-CCCCGGTCCGTTTGTTCTAT-3’；L2-R:5’-GCGAGTTGGATAGCCCGAGC-3’
L2-F:5’-TCTGGACGATTGGGCGACTT-3’；L3-R:5’-ACTTAGTCCGTTTCTCGGCTAT-3’
L3-F:5’-TAGACGCCAACTACGCTGAC-3’；L4-R:5’-AGGTTCCAACTGCTCTTACTGT-3’
(2) pcr amplification condition: with two-way expression vector p-T1 (4CL-CHS), p-T2 (CHI-FSII), p-T3 (PAL-C4H) template respectively, 94 DEG C of sex change 5 minutes, " 94 degree of sex change 30 seconds, 58 DEG C annealing 30 seconds, 72 DEG C extend 2 points 30 seconds " 34 circulations afterwards, 72 DEG C extend 10min, 4 DEG C of insulations.
(3) agarose gel electrophoresis purifying object product, result is as shown in Figure 18,19.
The conversion of the two-way expression vector p-T1 (4CL-CHS) of embodiment 8, p-T2 (CHI-FSII), p-T3 (PAL-C4H)
(1) activate W303 yeast saccharomyces cerevisiae, from W303 flat board, picking list bacterium colony spends the night and shakes training 12h in YPAD substratum (1% yeast extract, 2% peptone, 2% glucose, 50mg VITAMIN B4).
(2) OD600 of bacterium liquid is cultivated with spectrophotometric measurement.
(3) be worth for 0.2OD is transferred in the fresh YPAD substratum of 50ml with initial OD 600.
(4) activation allows yeast rise in value for two generations for 4-5 hour, and now bacterium liquid OD600 value is at 0.8-0.9.
(5) 3000g5min collects thalline, and 25mlddH2O washes twice.
(6) 1ml water is resuspended in aseptic 1.5ml centrifuge tube.
(7) the centrifugal 30s of 13000rpm collects thalline.
(8) the resuspended packing of 1ml water often pipe 100ul be used for transform, the bacterium liquid of packing centrifugal 20s on palm whizzer is abandoned and resets and add transformation system.
(9) transformation system:
| PEG3350 (50% (W/V filtration sterilization)) | 240ul |
| LiAc 1.0M (filtration sterilization) | 36ul |
| SSDNA(2.0mg/ml) | 50ul |
| Cotransformation DNA fragmentation and water | 34ul |
| Total | 360ul |
Fragment needed for cotransformation, adds 400ng by each fragment and mixes.
(10) transformation system is placed in 42 DEG C of water-bath heat shocks and is coated with CM-HIS solid medium (8g/L His Minus Media, 2% glucose, 1.5% agar powder) in 40 minutes.
(11) be placed in 30 DEG C of constant incubators cultivations and obtain recombinant Saccharomyces cerevisiae W303-1 in 2 ~ 3 days.
Checking after the two-way expression vector p-T1 (4CL-CHS) of embodiment 9, p-T2 (CHI-FSII), p-T3 (PAL-C4H) transform
(1) picking list bacterium colony 30 DEG C of 200rpm on the CM-HIS liquid nutrient medium of 5ml cultivate 12 ~ 16 hours.
(2) extract test kit with Yeast genome and extract recombinant Saccharomyces cerevisiae W303-1 genome.
(3) with the recombinant Saccharomyces cerevisiae W303-1 genome extracted for template PCR checking.
(a) design of primers
Co-F1：5’-AGGCAAGATAAACGAAGGCAAAG-3’；Co-R1：5’-GACACTGTTAGAATGAAGTTCCCAAA-3’
Co-F2：5’-TGCTACTAAGGCTATCAAGGAATGG-3’；Co-R2：5’-TGGTGTTTGGAAGGCTCACG-3’
Co-F3：5’-AACTGTTCTCAAATCTTGGGTTCTACT-3’；Co-R3：5’-CATCTTGGACGGTTCTGACTACG-3’
Co-F4：5’-AATACAACTACGGTGACTTCATCCC-3’；Co-R4：5’-GGCTCCCCTTAGACAAATACG-3’
B () PCR draws together increasing condition: polysaccharase is Easy Taq polysaccharase, take pastoris genomic dna as template, 94 DEG C of sex change 5 minutes, afterwards " 94 degree of sex change, 30 seconds, 58 DEG C annealing 30 seconds, 72 DEG C extend 1 point 15 seconds " 34 circulations, 72 DEG C extend 10min, 4 DEG C of insulations.
C () PCR terminates agarose gel electrophoresis checking, result as shown in figure 20.
Above preferred embodiment of the present invention has been described in detail, but described content being only preferred embodiment of the present invention, can not being considered to for limiting practical range of the present invention.All equalizations done according to the present patent application scope change and improve, and all should still belong within patent covering scope of the present invention.
Claims (9)
1. multiple foreign gene is cloned into the method for microbial genome by one kind simultaneously, it is characterized in that: multiple foreign gene is first imported multiple two-way expression vector, again multiple two-way expression vector is imported host microorganism simultaneously, obtain the recombinant microorganism of expressing multiple foreign gene;
Described each two-way expression vector includes a two-way terminator and a bidirectional promoter; Each two-way expression vector imports two foreign genes;
Except first two-way expression vector and last two-way expression vector, 3 ' end of other each two-way expression vectors and 5 ' end of the two-way expression vector of the next one have identical homology arm, 5 ' end of described first two-way expression vector has homology bracelet, 3 ' end of last two-way expression vector described has homology arm second, described homology bracelet and described homology arm second all can with the genome generation homologous recombination of host microorganism.
2. the method for claim 1, is characterized in that: described host microorganism is yeast; Be preferably yeast saccharomyces cerevisiae, be more preferably yeast saccharomyces cerevisiae W303.
3. the method for claim 1, it is characterized in that: described two-way expression vector has three, first two-way expression vector comprises the two-way terminator T1 as shown in SEQ ID NO:1 in sequence table, and the bidirectional promoter P1 shown in SEQ ID NO:4 in sequence table; Second two-way expression vector comprises the two-way terminator T2 as shown in SEQ ID NO:2 in sequence table, and the bidirectional promoter P2 shown in SEQ ID NO:5 in sequence table; 3rd two-way expression vector comprises the two-way terminator T3 as shown in SEQ ID NO:3 in sequence table, and the bidirectional promoter P1 shown in SEQ ID NO:4 in sequence table.
4. the method as described in any one of claim 1-3, it is characterized in that: described multiple foreign gene is 4CL, CHS, CHI, FSII, PAL, C4H gene, its gene order is successively as shown in sequence table SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11.
5. a DNA fragmentation, its nucleotides sequence is classified as any one all or part of of SEQ ID NO:1, SEQ ID NO:2 in sequence table, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5.
6. carry and express the recombinant expression vector of DNA fragmentation described in any one that nucleotides sequence is classified as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5 in sequence table.
7. recombinant expression vector according to claim 6 described in, is characterized in that:
Described recombinant expression vector is connected on pUC57-Kan plasmid by DNA fragmentation described in SEQ ID NO:1 in sequence table or SEQ ID NO:2 or SEQ ID NO:3 and obtains;
Or described recombinant expression vector is connected on PMD18-T plasmid by DNA fragmentation described in SEQ ID NO:4 in sequence table or SEQ ID NO:5 and obtains.
8. the first two-way expression vector obtained in method as claimed in claim 3 or the second two-way expression vector or the 3rd two-way expression vector.
9. the recombinant microorganism that method obtains according to any one of claim 1-4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410621447.0A CN104404077B (en) | 2014-11-06 | 2014-11-06 | A method of multiple foreign genes are cloned into microbial genome simultaneously |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410621447.0A CN104404077B (en) | 2014-11-06 | 2014-11-06 | A method of multiple foreign genes are cloned into microbial genome simultaneously |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104404077A true CN104404077A (en) | 2015-03-11 |
| CN104404077B CN104404077B (en) | 2018-11-09 |
Family
ID=52641743
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410621447.0A Active CN104404077B (en) | 2014-11-06 | 2014-11-06 | A method of multiple foreign genes are cloned into microbial genome simultaneously |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104404077B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636156A (en) * | 2016-12-26 | 2017-05-10 | 齐鲁工业大学 | Engineering bacterium capable of co-producing long-chain dicarboxylic acid and 1,3-propylene glycol and building method thereof |
| CN107586795A (en) * | 2017-10-10 | 2018-01-16 | 嘉兴欣贝莱生物科技有限公司 | A kind of method of fermentation by saccharomyces cerevisiae production phloretin |
| WO2018205977A1 (en) * | 2017-05-11 | 2018-11-15 | 杭州菁因康生物科技有限公司 | Efficient genetic engineering vector |
| CN112481178A (en) * | 2020-11-30 | 2021-03-12 | 上海交通大学 | Construction of amino bisdemethoxycurcumin high-yield strain and fermentation optimization method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329812A (en) * | 2011-09-21 | 2012-01-25 | 西南大学 | Multi-gene binary expression vector constructed by using homologous recombination and preparation method and application of multi-gene binary expression vector |
| CN102719481A (en) * | 2012-06-14 | 2012-10-10 | 中国科学院微生物研究所 | Method for simultaneously transferring multiple genes into microbial genome |
| CN103820488A (en) * | 2014-03-05 | 2014-05-28 | 怀化学院 | Novel method for co-transformation of pichia pastoris through multiple genes |
-
2014
- 2014-11-06 CN CN201410621447.0A patent/CN104404077B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329812A (en) * | 2011-09-21 | 2012-01-25 | 西南大学 | Multi-gene binary expression vector constructed by using homologous recombination and preparation method and application of multi-gene binary expression vector |
| CN102719481A (en) * | 2012-06-14 | 2012-10-10 | 中国科学院微生物研究所 | Method for simultaneously transferring multiple genes into microbial genome |
| CN103820488A (en) * | 2014-03-05 | 2014-05-28 | 怀化学院 | Novel method for co-transformation of pichia pastoris through multiple genes |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636156A (en) * | 2016-12-26 | 2017-05-10 | 齐鲁工业大学 | Engineering bacterium capable of co-producing long-chain dicarboxylic acid and 1,3-propylene glycol and building method thereof |
| CN106636156B (en) * | 2016-12-26 | 2021-03-19 | 齐鲁工业大学 | Engineering bacterium for co-producing long-chain dicarboxylic acid and 1, 3-propylene glycol and construction method thereof |
| WO2018205977A1 (en) * | 2017-05-11 | 2018-11-15 | 杭州菁因康生物科技有限公司 | Efficient genetic engineering vector |
| CN108866050A (en) * | 2017-05-11 | 2018-11-23 | 杭州菁因康生物科技有限公司 | A kind of efficient engineering carrier |
| CN108866050B (en) * | 2017-05-11 | 2022-12-27 | 杭州菁因康生物科技有限公司 | Efficient genetic engineering vector |
| CN107586795A (en) * | 2017-10-10 | 2018-01-16 | 嘉兴欣贝莱生物科技有限公司 | A kind of method of fermentation by saccharomyces cerevisiae production phloretin |
| CN112481178A (en) * | 2020-11-30 | 2021-03-12 | 上海交通大学 | Construction of amino bisdemethoxycurcumin high-yield strain and fermentation optimization method thereof |
| CN112481178B (en) * | 2020-11-30 | 2022-07-26 | 上海交通大学 | Construction of amino bisdemethoxycurcumin high-yield strain and fermentation optimization method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104404077B (en) | 2018-11-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104404077A (en) | Method for simultaneously cloning multiple exogenous genes to microbial genome | |
| CN101368188B (en) | Quick efficient plant manpower fine RNA expression vector construction method | |
| CN102732517B (en) | PCR (polymerase chain reaction) walking technology and kit used in same | |
| CN107858353A (en) | A kind of angstrom moral bacterium FS110 glutathione sulfurtransferase gene GliG promoters and its application | |
| CN104789556A (en) | Method of carrying out rapid and efficient DNA combination and evolution based on synthesis of single-chain DNA library | |
| CN100460515C (en) | Improved overlap extension PCR process and mutation gene obtained thereby | |
| CN109735537A (en) | A kind of myrothecium roidium A553 trichothecene synthase gene Tri5 promoter and its application | |
| CN108085308A (en) | A kind of recombination engineering that can improve thermostable lipase yield and its construction method and application | |
| CN105039382A (en) | Construction method of enramycin high-yielding strain and related gene | |
| CN104212757A (en) | Method for high-efficiently producing L-theanine through production of coli [gamma]-glutamylmethylamine synthetase with escherichia coli | |
| Liu et al. | Establishment of a genetic transformation system and its application in Thermoanaerobacter tengcongensis | |
| CN104673819A (en) | Vector for knocking out L-lactic dehydrogenase 1 gene and construction method of vector | |
| CN103131718B (en) | The clone of the new hypertonicity-resistant functional gene CgHog1 of source Candida glycerolgenesis and its application | |
| CN104561082B (en) | One plant of yeast Candida jeffriesii that can utilize xylose expression system | |
| CN104805508B (en) | A kind of method based on synthesizing single-stranded DNA library evolution metabolic pathway | |
| WO2020124831A1 (en) | Method for constructing high-efficiency bacillus subtilis promoters | |
| CN104212827B (en) | It is independent of the rapid molecular cloning process of bioengineered enzyme | |
| CN103397029B (en) | PCR walking method, and primer and kit thereof | |
| CN105624144B (en) | The construction method of saccharomyces cerevisiae ring chromosome | |
| CN108611360A (en) | A kind of production lycopene hydrogenlike silicon ion engineered strain and its construction method | |
| CN107267541A (en) | Utilize method of the galT gene deletion strains by fermenting and producing L amino acid | |
| CN105950613A (en) | Method for rapidly assembling non-phosphorylated DNA (deoxyribonucleic acid) fragments in vitro | |
| CN113186327A (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes FC89 strain, construction method and application thereof | |
| CN105802954A (en) | Method for rapidly and seamlessly assembling DNA in vitro on basis of heat-proof DNA polymerase and ligase | |
| CN107384923B (en) | Promoter pYLG and application thereof in construction of candida tropicalis with high yield of long-chain dicarboxylic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211208 Address after: 300308 1-201-a218, building 8, Huiying Industrial Park, No. 86, Zhonghuan West Road, pilot free trade zone (Airport Economic Zone), Binhai New Area, Tianjin Patentee after: Tiangong Biotechnology (Tianjin) Co.,Ltd. Address before: No.32, Xiqi Road, Airport Economic Zone, Binhai New Area, Tianjin 300308 Patentee before: TIANJIN INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY, CHINESE ACADEMY OF SCIENCES |