Construction of a new gene integration platform system for theSynechococcus sp. PCC7942

Zhang Jun; Wu Qiao-juan; Lou Shi-lin

doi:10.1007/BF02849078

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Construction of a new gene integration platform system for theSynechococcus sp. PCC7942

Updated：2023-05-16

- Construction of a new gene integration platform system for theSynechococcus sp. PCC7942
- Journal of Oceanology and Limnology Vol. 16, Issue 1, Pages: 40-46(1998)
- Affiliations：
  
  Biology Department Xiamen University
- Author bio：
- Funds：
- DOI：10.1007/BF02849078
  CLC：
- Published：1998
- Accepted：
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Jun, Z., Qiao-juan, W. & Shi-lin, L. Construction of a new gene integration platform system for theSynechococcus sp. PCC7942., Chin. J. Ocean. Limnol. 16, (Suppl 1), 40–46 (1998). https://doi.org/10.1007/BF02849078
DOI：

Jun, Z., Qiao-juan, W. & Shi-lin, L. Construction of a new gene integration platform system for theSynechococcus sp. PCC7942., Chin. J. Ocean. Limnol. 16, (Suppl 1), 40–46 (1998). https://doi.org/10.1007/BF02849078 DOI：

摘要

Abstract

According to the known sequence of iron stress-induced gene (isiAB operon)

we cloned its 1.5 kb fragment by PCR

and used this fragment as integration homologous fragment. After several steps of subcloning donor DNA into theisiAB fragment

a donor plasmid pZL which could be integrated into the chromosomal DNA ofSynechococcus sp. PCC7942 was constructed. In order to express the heterologous gene at a high level through the integration platform system

we constructed the donor DNA by the following steps. We cloned the strong promoter (240 bp) of heat shock genegroESL operon fromSynechococcus sp. PCC7942 by PCR. Then subcloned the multiple cloning sites (MCS)

rbcS polyA into the downstream of thegroESL promoter. The kanamycin resistance gene

as the marker gene

was also subcloned into the donor DNA. Thus

in the donor plasmid pZL

the integration homologous fragment and several expression elements

such asgroESL promoter

MCS

rbcS polyA terminator and kanamycin resistance gene

were all included. After naturally transformed and introduced the donor plasmid pZL intoSynechococcus sp. PCC7942

as in the pZL

the donor DNA sequence is flanked by two DNA fragments (0.4 kb and 0.7 kb) homologous to theisiAB fragment ofSynechococcus sp. PCC7942

the homologous DNA can recombine with the chromosomal DNA. After screening by kanamycin

the transformants which integrated the heterologous DNA were selected. The efficiency of transformation is about 1×10−6. By southern blot analysis

it was confirmed that the donor DNA had been integrated into the chromosomal DNA ofSynechococcus sp. PCC7942

located on the site of theisiAB gene

and can be replicated with the chromosomal DNA.

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references

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