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Universal plasmid, construction method of universal plasmid and novel method for expressing exogenous genes by synechocystis

A Synechocystis and plasmid technology, applied in the field of genetic engineering, can solve the problems of weakened start-up ability and dependence

Pending Publication Date: 2021-04-13
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the promoter is dependent on light, and its ability to start is weakened in the absence of light

Method used

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  • Universal plasmid, construction method of universal plasmid and novel method for expressing exogenous genes by synechocystis
  • Universal plasmid, construction method of universal plasmid and novel method for expressing exogenous genes by synechocystis
  • Universal plasmid, construction method of universal plasmid and novel method for expressing exogenous genes by synechocystis

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Experimental program
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Effect test

Embodiment 1

[0052] Construction of plasmid P5ST1T2-downstream:

[0053] (1) Use SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing as upstream and downstream primers, use the wild Synechocystis PCC6803 genome as a template; use SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing as upper For downstream primers, the Genbank accession number is U02439.1 Escherichia coli as a template; with SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing as upstream and downstream primers, using wild Synechocystis sp. PCC6803 as a template, amplified by PCR The technology obtains the photosensitive promoter and the upstream arm gene sequence Promoter-up, the terminator T1T2 gene fragment and the downstream arm sequence downstream. The obtained sequence is shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 in the sequence listing; (2) Promoter-up of the light-sensitive promoter and upstream arm gene sequence obtained in step (1) Carry out ApaI enzyme digestion treatment, perform PSt I and Ba...

Embodiment 2

[0056] Construction of Universal Plasmid P5ST1T2Kana

[0057] (1) Treat the puc4K plasmid with the restriction endonuclease BamHI to recover the Kana resistance sequence, the sequence is shown in SEQ ID NO: 14 in the sequence listing; (2) use SEQ ID NO: 7 and SEQ ID NO in the sequence listing: 8 is the upstream and downstream primers, use the sequence recovered in step (1) as a template, and obtain the promoter and Kana resistance gene sequence by PCR amplification technology: Promote-Kana, add enzymes at the upstream and downstream ends of Promote-Kana respectively through primers Cutting sites BamHI and NheI, the obtained sequence is shown in SEQ ID NO: 15 in the sequence listing; using SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing as upstream and downstream primers, and the sequence recovered in step 2 is Template, obtain the terminator gene sequence by PCR amplification technology: terminator, respectively add enzyme cutting sites Nhe I and BamHI at the upstream a...

Embodiment 3

[0060] Construction of Recombinant Plasmid PSKC II Expressing Cellulase in Synechocystis sp.

[0061] Using SEQ ID NO: 17 and SEQ ID NO: 18 in the sequence listing as upstream and downstream primers, using the corresponding gene sequence of Trichoderma reesei (Trichoderma reesei QM9414) in the GenBank database as a template, the cellulase exonuclease CBH was obtained by PCR technology II gene fragment, and add homology arm sequences at both ends of the target gene through primers, the sequence is shown in SEQID NO: 19 in the sequence listing; the shuttle recombinant expression vector P5ST1T2Kana is treated with the restriction endonuclease Nhe I, and the obtained The gene fragment of cellulosic exonuclease CBH II undergoes homologous recombination under the action of recombinase to obtain recombinant expression vector plasmid PSKC II.

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Abstract

According to the invention, a universal plasmid for promoting expression of a target gene in synechocystis PCC 6803 by using a Kana resistance gene promoter is constructed through a genetic engineering method; on the basis that the plasmid uses a psbA2 gene initiation codon ATG first 510 bp fragment of the synechocystis PCC 6803 as a homologous recombination upstream arm and a psbA2 gene ORF fragment as a downstream arm, the target gene is inserted between the Kana resistance gene and a terminator to obtain the universal plasmid P5ST1T2Kana; and the Kana resistance gene promoter is used for promoting the tandem expression of the kana resistance gene and the target gene in the synechocystis. Meanwhile, a synechocystis PCC 6803 engineering algal strain PSKC II capable of promoting the tandem expression of the Kana resistance gene and a cellulase CBHII gene by the Kana gene promoter is constructed by taking the universal plasmid as a vector, and a new means of heterologous expression of cellulase in the synechocystis is realized.

Description

technical field [0001] The present invention relates to a universal plasmid for promoting the expression of a target gene in Synechocystis spp. The method belongs to the field of genetic engineering. Background technique [0002] A plasmid is a closed circular double-stranded DNA molecule that has the ability to replicate autonomously, allowing it to maintain a constant copy number in progeny cells and express the genetic information it carries. Plasmids are found in all bacterial taxa and are DNA molecules that self-replicate independently of the bacterial extrachromosomal. Bacterial plasmids are commonly used vectors in recombinant DNA technology. A vector is a tool for delivering a useful foreign gene into recipient cells for proliferation and expression through genetic engineering. A certain target gene fragment is recombined into a plasmid to form a recombinant gene or a recombinant. Then the recombinant is transformed into the recipient cell through microbiological...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/66C12N15/65C12N15/56C12N1/21C12R1/01
CPCC12N15/74C12N15/66C12N15/65C12N9/2437
Inventor 戴玉杰宁玉林吕和鑫张黎明满淑丽贾士儒
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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