Analysis of the leakage of gene repression by an artificial TetR-regulated promoter in cyanobacteria

BMC Research Notes

Peter Lindblad²  U. Helena Danielson¹  Christian Seeger¹  Hsin-Ho Huang² 

[1] Department of Chemistry - BMC, Uppsala University, Uppsala, 751 23, Sweden;Department of Chemistry - Ångström, Science for Life Laboratory, Microbial Chemistry, Uppsala University, Uppsala, 751 20, Sweden

关键词: EPBD model;    DNA breathing dynamics;    SPR;    Promoter;    TetR binding;    Synechocystis;    Cyanobacteria;   

Others  :  1230102
DOI  :  10.1186/s13104-015-1425-0

 received in 2014-06-29, accepted in 2015-09-08,  发布年份 2015

【 摘 要 】

Background

There is a need for strong and tightly regulated promoters to construct more reliable and predictable genetic modules for synthetic biology and metabolic engineering. For this reason we have previously constructed a TetR regulated L promoter library for the cyanobacterium Synechocystis PCC 6803. In addition to the L03 promoter showing wide dynamic range of transcriptional regulation, we observed the L09 promoter as unique in high leaky gene expression under repressed conditions. In the present study, we attempted to identify the cause of L09 promoter leakage. TetR binding to the promoter was studied by theoretical simulations of DNA breathing dynamics and by surface plasmon resonance (SPR) biosensor technology to analyze the kinetics of the DNA–protein interactions.

Results

DNA breathing dynamics of a promoter was computed with the extended nonlinear Peyrard–Bishop–Dauxois mesoscopic model to yield a DNA opening probability profile at a single nucleotide resolution. The L09 promoter was compared to the L10, L11, and L12 promoters that were point-mutated and different in repressed promoter strength. The difference between DNA opening probability profiles is trivial on the TetR binding site. Furthermore, the kinetic rate constants of TetR binding, as measured by SPR biosensor technology, to the respective promoters are practically identical. This suggests that a trivial difference in probability as low as 1 × 10 ⁻⁴cannot lead to detectable variations in the DNA–protein interactions. Higher probability at the downstream region of transcription start site of the L09 promoter compared to the L10, L11, and L12 promoters was observed. Having practically the same kinetics of binding to TetR, the leakage problem of the L09 promoter might be due to enhanced RNA Polymerase (RNAP)-promoter interactions in the downstream region.

Conclusions

Both theoretical and experimental analyses of the L09 promoter’s leakage problem exclude a mechanism of reduced TetR binding but instead suggest enhanced RNAP binding. These results assist in creating more tightly regulated promoters for realizing synthetic biology and metabolic engineering in biotechnological applications.

【 授权许可】

   
2015 Huang et al.

【 图 表 】

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