Artificial chromosome as well as construction method and application thereof

Abstract

The invention belongs to the technical field of biology, and provides an artificial chromosome as well as a construction method and application thereof. The artificial chromosome comprises an initial artificial chromosome and a functional gene which are connected; the functional genes comprise a Cas9 gene, a Ura selection marker gene, a gRNA recognition sequence, a homologous sequence 1 and a fragment homologous sequence. The construction method comprises the following steps: connecting a functional gene to an initial artificial chromosome in an enzyme digestion and enzyme ligation manner; transforming a connection product into escherichia coli, and picking positive clones from a chloramphenicol resistant plate; and intracellular assembly of the large DNA fragment: introducing the artificial chromosome and the large DNA fragment to be assembled into a yeast cell, and completing assembly through intracellular homologous recombination. The invention also discloses application of the artificial chromosome in gene cloning and DNA synthesis. According to the artificial chromosome disclosed by the invention, the synthesis efficiency of large-fragment DNA can be improved, the problems of easy breakage and low efficiency in in-vitro operation are avoided, and a tool is provided for subsequent synthesis of a complete genome.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to an artificial chromosome and a construction method and application thereof. Background Art

[0002] Yeast artificial chromosome is a vector that exists in yeast cells and can clone DNA fragments up to several hundred kb. It contains the necessary telomeres, centromeres and replication initiation sequences in yeast cells and is an object with genetic properties in cells. In synthetic biology, yeast artificial chromosomes are mainly used to construct large-fragment DNA libraries, especially for constructing genomic libraries of higher eukaryotes, and are not used for conventional gene cloning.

[0003] The synthesis of large DNA fragments is a key step in achieving the synthesis of a complete genome of an organism, and the stable existence and proliferation of DNA fragments in the organism are necessary conditions. The use of yeast artificial chromosomes to synthesize target large DNA fragments is to introduce DNA fragments into microbial cells and connect them to the designated parts of yeast artificial chromosomes. DNA synthesis technology is a key technology in synthetic biology. In recent years, with the rapid development of DNA sequencing technology and DNA synthesis technology, the rapid development of synthetic biology, an emerging discipline that uses engineering methods to design and synthesize genes, biological elements, metabolic pathways and even genomes, has made the role of DNA synthesis technology more prominent. Although traditional small fragment synthesis and gene manipulation still play an important role in many aspects, they can no longer meet the needs of designing larger and larger manipulation units.

[0004] Currently, humans have begun to try to synthesize larger genomes of microorganisms and even mammals. The scale of synthetic genomes is huge, and conventional vectors are no longer able to load such large-scale genomes, let alone efficiently complete the assembly process. Therefore, in order to meet the needs of large-scale DNA assembly, a corresponding vector must be developed to achieve efficient and stable genome assembly. Summary of the invention

[0005] The present disclosure provides an artificial chromosome and a construction method and application thereof, so as to at least solve the above technical problems existing in the prior art.

[0006] According to the first aspect of the present disclosure, an artificial chromosome pCU is provided, comprising an initial artificial chromosome, and a functional gene connected to the initial artificial chromosome; the functional gene comprises a Cas9 gene, an auxotrophic screening marker gene, a gRNA recognition sequence, a homologous sequence 1 and a fragment homologous sequence.

[0007] In one embodiment, the nucleic acid sequence of the functional gene is as shown in SEQ ID NO:1.

[0008] Specifically, the full length of the nucleic acid sequence of the functional gene is 6295bp, of which 1-1180bp is the fragment homologous sequence, 1181-5280bp is the Cas9 gene, 5281-6179bp is the nutritional deficiency screening marker gene, 6201-6244bp is the gRNA recognition sequence, and 6245-6295bp is the homologous sequence 1.

[0009] In one embodiment, the auxotrophic selection marker gene is the Ura selection marker gene.

[0010] In one embodiment, the initial artificial chromosome is pcci-2μ.

[0011] In one embodiment, the initial artificial chromosome and the functional gene are connected by Gibson connection.

[0012] According to the second aspect of the present disclosure, a method for constructing the above-mentioned artificial chromosome pCU is provided, comprising the following steps: connecting the functional gene to the initial artificial chromosome by enzyme cutting and enzyme ligation; then transforming the connection product into Escherichia coli, and picking positive clones on a chloramphenicol resistance plate to obtain the artificial chromosome pCU.

[0013] In one embodiment, the functional gene is connected to the site after the BamHI restriction site of the initial artificial chromosome by enzyme cleavage and enzyme ligation.

[0014] In one embodiment, after the ligation product is transformed into the E. coli, the transformed E. coli is spread on the chloramphenicol resistance plate for culture, and then the positive clone is picked.

[0015] According to the third aspect of the present disclosure, a method for intracellular assembly of large DNA fragments using the artificial chromosome pCU is provided, comprising the following steps: introducing the artificial chromosome pCU and the large DNA fragment to be assembled into yeast cells, and completing the assembly through intracellular homologous recombination.

[0016] In one embodiment, the length of the large DNA fragment to be assembled is 100 to 800 Kb.

[0017] In a preferred embodiment, the length of the large DNA fragment to be assembled is 200 Kb.

[0018] In a preferred embodiment, the large DNA fragment to be assembled is assembled from 20 10Kb DNA fragments in the cell; in two adjacent DNA fragments, the 3' end of the former DNA fragment and the 5' end of the latter DNA fragment both contain 300 to 700 bp of identical sequence, ensuring efficient homologous recombination in the cell.

[0019] In one embodiment, the homologous sequence 1 of the artificial chromosome pCU is homologous to a partial sequence at either the N-terminus or the C-terminus of the large DNA fragment to be assembled, and the fragment homologous sequence of the artificial chromosome pCU is homologous to a partial sequence at the other of the N-terminus and the C-terminus of the large DNA fragment to be assembled.

[0020] Specifically, if the homologous sequence 1 of the artificial chromosome pCU is homologous to a partial sequence at the N-terminus of the large DNA fragment to be assembled, then the fragment homologous sequence of the artificial chromosome pCU is homologous to a partial sequence at the C-terminus of the large DNA fragment to be assembled; conversely, if the homologous sequence 1 is homologous to a partial sequence at the C-terminus of the large DNA fragment to be assembled, then the fragment homologous sequence is homologous to a partial sequence at the N-terminus of the large DNA fragment to be assembled.

[0021] In one embodiment, the artificial chromosome pCU and the large DNA fragment to be assembled are introduced into the yeast cells by lithium acetate transformation method.

[0022] According to a fourth aspect of the present disclosure, there is provided application of the artificial chromosome pCU in gene cloning and DNA synthesis.

[0023] According to an implementable embodiment of the present disclosure, at least the following beneficial effects are achieved:

[0024] The present invention designs and transforms artificial chromosomes and realizes the one-time assembly of large DNA fragments in yeast cells by leveraging the efficient homologous recombination ability in yeast cells, thereby improving the synthesis efficiency of large DNA fragments, avoiding the problems of easy breakage and low efficiency in in vitro operations, and providing a tool for the subsequent synthesis of larger-scale complete genomes.

[0025] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present disclosure, nor is it intended to limit the scope of the present disclosure. Other features of the present disclosure will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The above and other objects, features and advantages of the exemplary embodiments of the present disclosure will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, in which:

[0027] In the drawings, the same or corresponding reference numerals represent the same or corresponding parts.

[0028] Figure 1 A map of the artificial chromosome pCU constructed in Example 1 of the present disclosure is shown;

[0029] Figure 2 The figure shows the agarose gel electrophoresis result of 20 DNA fragments amplified in Example 2 of the present disclosure;

[0030] Figure 3 The agarose gel electrophoresis result of the colony PCR product in Example 2 of the present disclosure is shown;

[0031] Figure 4 The figure shows the pulse field electrophoresis result in Example 2 of the present disclosure. DETAILED DESCRIPTION

[0032] In order to make the purpose, features, and advantages of the present disclosure more obvious and easy to understand, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.

[0033] Example 1

[0034] In this embodiment, an artificial chromosome pCU was constructed, and the specific process was as follows:

[0035]

[0036] (2) The synthesized functional gene was connected to the site behind the BamHI restriction site of the initial artificial chromosome pcci-2μ by enzyme digestion and enzyme ligation; the ligation product was transformed into Escherichia coli EPI300 by chemical transformation, and then the transformed Escherichia coli EPI300 was spread on a chloramphenicol resistant plate for culture, and positive clones were picked on the cultured plate to obtain the artificial chromosome pCU. The length of the artificial chromosome pCU is 18373 bp, and the sequencing results are consistent with the design. The map of the constructed artificial chromosome pCU is shown in the figure. Figure 1 shown.

[0037] Example 2

[0038] This example uses the artificial chromosome pCU constructed in Example 1 to perform intracellular assembly of large DNA fragments. The specific process is as follows:

[0039] The assembled fragments come from the first 200Kb of the Bacillus subtilis 168 genome (RefSeq: GCF_000009045.1), and are assembled into a 200Kb large DNA fragment in the cell from 20 10Kb fragments. In two adjacent 10Kb DNA fragments, the 3' end of the former DNA fragment and the 5' end of the latter DNA fragment both contain 300 to 700bp of identical sequences, ensuring efficient homologous recombination in the cell.

[0040] (1) The genome of Bacillus subtilis 168 was extracted, and 20 10 Kb DNA fragments were amplified by PCR using the genome of Bacillus subtilis 168 as a template. The corresponding 20 pairs of primers were numbered p1-20-F / R, and the primer sequences were as follows:

[0041] p1-F, SEQ ID NO:2: TCGACAACATTTTCACATTACCAACCCCTGT; p1-R, SEQ ID NO:3: CGCTCCCCACGCTTTCGCTCCT;

[0042] p2-F, SEQ ID NO:4: GGGACTGAGACACGGCCCAGACTCC;

[0043] p2-R, SEQ ID NO:5: CATCTTTCACCGATTCGCACCAACCA;

[0044] p3-F, SEQ ID NO:6:TCTATCGGAGCATAATGGTCGTATTGTAGCC;

[0045] p3-R,SEQ ID NO:7:TCCTCTTCTGCACTCAAGTTCCCCAGTTT;

[0046] p4-F,SEQ ID NO:8:AGGCGACGATGCGTAGCCGACC;

[0047] p4-R,SEQ ID NO:9:GGTTCCCTGACTCTATCCGTTTACAGTTTCG;

[0048] p5-F,SEQ ID NO:10:CTTTGACCTTTACGGGAACAGCAGAACG;

[0049] p5-R,SEQ ID NO:11:CACGCCCCTCGACCACAATGATC;

[0050] p6-F,SEQ ID NO:12:CCGAATGCCAAAGTCATCGCTGTG;

[0051] p6-R,SEQ ID NO:13:CGGTTTACTCAAAGAAGCCTCACAAGCCT;

[0052] p7-F,SEQ ID NO:14:TTTACGACGACCTTGACCTTCCGACTG;

[0053] p7-R,SEQ ID NO:15:GCTTGACCAACGGGCCAAACAAATATG;

[0054] p8-F,SEQ ID NO:16:AAAAGCTAAAGACCGTCCGCAAGCCA;

[0055] p8-R,SEQ ID NO:17:CACAAGATAGTAAGTAAAATCATCTCCTAAT TCTCCTGAAA;

[0056] p9-F,SEQ ID NO:18:ACCAAAGGTCATTGCGACAGGAGGC;

[0057] p9-R,SEQ ID NO:19:CCCAGTCTTACAGGCAGGTTACCCACG;

[0058] p10-F,SEQ ID NO:20:CTTCACTGAGTCAACGAGTTATAATAATAAA GTCGCTTGAAC;

[0059] p10-R,SEQ ID NO:21:CAGGGTAGTATCCCACCGATGCCTCCAC;

[0060] p11-F,SEQ ID NO:22:CCAAGCAGTTAGGCTGGGAAATAGGCA;

[0061] p11-R,SEQ ID NO:23:ATCATCGTACCGTCATCCAAGTAGGCAA;

[0062] p12-F,SEQ ID NO:24:GAATTGATCAGCCTGTTTTCCATTTCCG;

[0063] p12-R,SEQ ID NO:25:CACGACCGATTTTACCAACTTCACCCAT;

[0064] p13-F,SEQ ID NO:26:ACAGTTGAAGTGGCTTTCCGTTTAGGGG;

[0065] p13-R,SEQ ID NO:27:TGTGCTCTTGCAGGTTGTGACCGAT;

[0066] p14-F,SEQ ID NO:28:TTTTGCCTTTTGATGAACCACCTGGG;

[0067] p14-R,SEQ ID NO:29:CGCTCCGCTTTTAGTGCGAACGATA;

[0068] p15-F,SEQ ID NO:30:GGATAAAACCATTACTGTTGTTGTTGAGACA TACA;

[0069] p15-R,SEQ ID NO:31:TCACCACGGCGAGGACCAAGC;

[0070] p16-F,SEQ ID NO:32:ACAGCATTCCAATAAAGGAGGGGACATC;

[0071] p16-R, SEQ ID NO:33:TTTTCAACTGACACCGGACCTAAAGCC;

[0072] p17-F, SEQ ID NO:34: ACACTGTAATGAACGACTGTATAATTGTATTGCTTTGCG;

[0073] p17-R, SEQ ID NO:35: GACAGTGCCCAGATCGTTGCGCCTTT;

[0074] p18-F, SEQ ID NO:36: GTATAGGGCTGACGCCTGCCCGGTGCT;

[0075] p18-R, SEQ ID NO:37: CCCCATTGCTCAAATGGTGAGGAGGTGGTA;

[0076] p19-F, SEQ ID NO:38: CCAAAGTATACGTTAAGTGGAAAAGGATGT GGAG;

[0077] p19-R, SEQ ID NO:39: AAGTGATGACAGGTCTTCTTCGTCAGGAAATG; p20-F, SEQ ID NO:40: TCGATTGAAGCAGTTTGTTCTTCGTAAGGTG; p20-R, SEQ ID NO:41: CGCATAACAACAGGCTGCTCATCCG;

[0078] PCR reaction system: 10 μL of 1× PrimeSTAR GXL Buffer, 4 μL of 2.5 mM dNTP Mixture, 1 μL of upstream and downstream primers, 1 μL of template, 1 μL of PrimeSTAR GXL DNA Polymerase, and add water to make up to 50 μL.

[0079] PCR reaction conditions: 98°C, 10s; 68°C, 10min, 30 cycles; stored at 4°C.

[0080] After PCR, agarose gel electrophoresis was performed to verify the DNA band size. Figure 2 shown. Figure 2 The results showed that the sizes of the 20 DNA fragments were all within 10 Kb, indicating successful amplification.

[0081] (2) Twenty DNA fragments containing homology arms and the artificial chromosome pCU were co-transformed into yeast cells using the Saccharomyces cerevisiae lithium acetate transformation method.

[0082] Use an inoculation loop to dip the preserved BY4741 glycerol culture, inoculate it into solid YPD medium by streaking, and culture it at 30°C overnight;

[0083] Pick a single colony and inoculate it into 50 mL of YPD liquid medium, culture it at 200 rpm and 30°C overnight;

[0084] Initial OD 600 Transfer to a new 50 mL YPD liquid medium and culture at 200 rpm and 30°C until the OD is 0.1. 600 Reach 0.5; take 1mL of bacterial solution, centrifuge at 4000rpm for 1min, harvest the cells; resuspend with 1mL of sterile water and wash the cells, centrifuge at 4000rpm for 1min, harvest the cells; resuspend the cells with 1mL, 0.1M LioAc and place on ice.

[0085] First, boil the ssDNA at 101°C for 10 min and immediately cool it on ice. Then prepare the transformation system: 620 μL of 50% PEG3350, 90 μL of 1M LioAc, and 40 μL of the 10 mg / mL boiled ssDNA mentioned above, and mix them evenly.

[0086] The cells resuspended in the above LioAc were centrifuged at 4000rpm for 1min, and the supernatant was discarded; 5μL of artificial chromosome pCU and 5μL of 20 DNA fragments were aspirated and mixed evenly with the cells, then spirally added to the above transformation system, inverted evenly, and incubated at 200rpm and 30℃ for 30min; 90μL of DMSO was added and inverted evenly; heat shocked at 42℃ for 18min, centrifuged at 4000rpm for 2min, harvested the cells, added 400μL of 5mM CaCl2 solution, resuspended the cells, and allowed to stand for 5min; 100μL was spread on the SC-Ura screening plate, and single colonies were visible after incubation at 30℃ for 2-3 days.

[0087] After a single colony grows, pick half of the single colony and suspend it in 50 μL of 20mM NaOH. Perform three cycles in a PCR instrument (99°C, 5min; 4°C, 60s) to fully lyse the yeast cells. Then use 1 μL of the yeast cell lysate as a template for colony PCR reaction. The colony PCR primers are numbered p21-25-F / R, as follows:

[0088] p21-F, SEQ ID NO:42: ATCTTGACAGGTGTAAACTGGAAAGTGGAGC;

[0089] p21-R, SEQ ID NO:43: CCGCCAGACTGAATCACATCGGCTAAT;

[0090] p22-F, SEQ ID NO:44: GAGTATCATCGTCTGGGTGACACTGAAGCA;

[0091] p22-R, SEQ ID NO:45: TGTGACAGGGGCAACCAAGTTGGAATT;

[0092] p23-F, SEQ ID NO:46: TTCATCTGCCAATCCTGCGGCTACG;

[0093] p23-R, SEQ ID NO:47: TGTCAACCATATGCTCCAACAGTCTCGG;

[0094] p24-F, SEQ ID NO:48: GTTAGGCGAAGGGAGGACGAAAGGCA;

[0095] p24-R, SEQ ID NO:49: GGATCGGACATTCTGGTTCCAGCTACACT;

[0096] p25-F, SEQ ID NO:50: CAATGGCTGATGGACCAAGCGATAACA;

[0097] p25-R, SEQ ID NO:51: TGCGGAACACCCGTCACGACGTA;

[0098] PCR reaction system: 2 μL of 10× PCR buffer, 2 μL of 2.5 mM dNTPs, 1 μL of upstream and downstream primers, 1 μL of template, 0.25 μL of Taq DNA polymerase, and add water to 20 μL.

[0099] PCR reaction conditions: 95℃ pre-denaturation for 2-5min; 95℃ denaturation for 20s, 51-66℃ annealing for 20s, 72℃ extension for 4min, 30 cycles; 72℃ extension for 5min, 4℃ storage. The DNA band size was verified by agarose gel electrophoresis. The results are as follows: Figure 3 shown. Figure 3 It showed that the sizes of the five bands were consistent with expectations, indicating that the five colonies picked were all positive clones and that they had successfully performed intracellular assembly of large DNA fragments.

[0100] The artificial chromosomes were extracted from clones with the correct band size and verified by pulsed field electrophoresis. The artificial chromosomes were extracted using the Biomed DP104-01 yeast high-purity plasmid small-volume rapid extraction kit. The pulsed field electrophoresis conditions were as follows: 1% low-melting point agarose gel, 4.5 V / cm, 10°C, 42 h, switching time 99 s, and the results are shown in the figure. Figure 4 . Figure 4 It was shown that the large DNA fragment connected in the artificial chromosome was indeed 200Kb.

[0101] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps recorded in this disclosure can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solution of this disclosure can be achieved, and this document is not limited here.

[0102] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0103] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present disclosure, which should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. An artificial chromosome pCU, characterized in that The artificial chromosome pCU includes an initial artificial chromosome and a functional gene connected to the initial artificial chromosome; the functional gene includes a Cas9 gene, an auxotrophic screening marker gene, a gRNA recognition sequence, a homologous sequence 1 and a fragment homologous sequence.

2. The artificial chromosome pCU according to claim 1, characterized in that The nutritional deficiency screening marker gene is a Ura screening marker gene; Preferably, the initial artificial chromosome is pcci-2μ; Preferably, the initial artificial chromosome and the functional gene are connected by Gibson connection.

3. The artificial chromosome pCU according to claim 1, characterized in that The nucleic acid sequence of the functional gene is shown in SEQ ID NO: 1; the full length of the nucleic acid sequence of the functional gene is 6295 bp, wherein 1-1180 bp is the fragment homologous sequence, 1181-5280 bp is the Cas9 gene, 5281-6179 bp is the auxotrophic screening marker gene, 6201-6244 bp is the gRNA recognition sequence, and 6245-6295 bp is the homologous sequence 1.

4. The method for constructing the artificial chromosome pCU according to any one of claims 1 to 3, characterized in that: The following steps are involved: The functional gene is connected to the initial artificial chromosome by enzyme cutting and enzyme ligation; the connection product is then transformed into Escherichia coli, and positive clones are picked on a chloramphenicol resistance plate to obtain the artificial chromosome pCU.

5. The construction method according to claim 4, characterized in that: The functional gene is connected to the site behind the BamHI restriction site of the initial artificial chromosome by means of enzyme cutting and enzyme ligation.

6. A method for intracellular assembly of large DNA fragments using the artificial chromosome pCU according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: introducing the artificial chromosome pCU and the large DNA fragment to be assembled into yeast cells, and completing the assembly through intracellular homologous recombination.

7. The method according to claim 6, characterized in that The length of the large DNA fragment to be assembled is 100 to 800 Kb.

8. The method according to claim 6, characterized in that The homologous sequence 1 of the artificial chromosome pCU is homologous to a partial sequence at either the N-terminus or the C-terminus of the large DNA fragment to be assembled, and the fragment homologous sequence of the artificial chromosome pCU is homologous to a partial sequence at the other of the N-terminus and the C-terminus of the large DNA fragment to be assembled.

9. The method according to claim 6, characterized in that The artificial chromosome pCU and the large DNA fragment to be assembled are introduced into the yeast cells by lithium acetate transformation method.

10. Use of the artificial chromosome pCU according to any one of claims 1 to 3 in gene cloning and DNA synthesis.

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