Dataset Information

Construction and characterization of a synthetic Baculovirus-inducible 39K promoter.

ABSTRACT:

Background

Silkworm genetic engineering is widely used in gene function, silk engineering and disease-resistant engineering in most of Asia. Some of the earliest promoter elements are used to control the development of silkworm transgenic expression and gene therapy. However, the low expression and specificity of natural promoters limit the applications of genetic engineering. To construct a highly efficient synthetic inducible promoter in the Bombyx mori (Lepidoptera), we analyzed the regulatory elements and functional regions of the B. mori nucleopolyhedrovirus 39?K promoter.

Results

Truncated mutation analysis of the 39?K promoter showed that the transcriptional regulatory region spanning positions -?573 to -?274 and?+?1 to +?62 are essential for virus-inducible promoter activity. Further investigations using the electrophoretic mobility shift assay revealed that the baculovirus IE-1 protein binds to the 39?K promoter at the -?310 to -?355 region, and transcription activates the expression of 39?K promoter assay. Finally, we successfully constructed a synthetic inducible promoter that increased the virus-inducing activity of other promoters using the baculovirus-inducible transcriptional activation region that binds to specific core elements of 39?K (i.e., spanning the region -?310 to -?355).

Conclusions

In summary, we constructed a novel, synthetic, and highly efficient biological tool, namely, a virus-inducible 39?K promoter, which provides endless possibilities for future research on gene function, gene therapy, and pest control in genetic engineering.

SUBMITTER: Dong ZQ

PROVIDER: S-EPMC6280533 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Construction and characterization of a synthetic Baculovirus-inducible 39K promoter.

Dong Zhan-Qi ZQ Hu Zhi-Gang ZG Li Hai-Qing HQ Jiang Ya-Ming YM Cao Ming-Ya MY Chen Peng P Lu Cheng C Pan Min-Hui MH

Journal of biological engineering 20181204

<h4>Background</h4>Silkworm genetic engineering is widely used in gene function, silk engineering and disease-resistant engineering in most of Asia. Some of the earliest promoter elements are used to control the development of silkworm transgenic expression and gene therapy. However, the low expression and specificity of natural promoters limit the applications of genetic engineering. To construct a highly efficient synthetic inducible promoter in the <i>Bombyx mori</i> (Lepidoptera), we analyze ...[more]

PMID: 30534200

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Project description:BackgroundThe bottom-up programming of living organisms to implement novel user-defined biological capabilities is one of the main goals of synthetic biology. Currently, a predominant problem connected with the construction of even simple synthetic biological systems is the unpredictability of the genetic circuitry when assembled and incorporated in living cells. Copy number, transcriptional/translational demand and toxicity of the DNA-encoded functions are some of the major factors which may lead to cell overburdening and thus to nonlinear effects on system output. It is important to disclose the linearity working boundaries of engineered biological systems when dealing with such phenomena.ResultsThe output of an N-3-oxohexanoyl-L-homoserine lactone (HSL)-inducible RFP-expressing device was studied in Escherichia coli in different copy number contexts, ranging from 1 copy per cell (integrated in the genome) to hundreds (via multicopy plasmids). The system is composed by a luxR constitutive expression cassette and a RFP gene regulated by the luxI promoter, which is activated by the HSL-LuxR complex. System output, in terms of promoter activity as a function of HSL concentration, was assessed relative to the one of a reference promoter in identical conditions by using the Relative Promoter Units (RPU) approach. Nonlinear effects were observed in the maximum activity, which is identical in single and low copy conditions, while it decreases for higher copy number conditions. In order to properly compare the luxI promoter strength among all the conditions, a mathematical modeling approach was used to relate the promoter activity to the estimated HSL-LuxR complex concentration, which is the actual activator of transcription. During model fitting, a correlation between the copy number and the dissociation constant of HSL-LuxR complex and luxI promoter was observed.ConclusionsEven in a simple inducible system, nonlinear effects are observed and non-trivial data processing is necessary to fully characterize its operation. The in-depth analysis of model systems like this can contribute to the advances in the synthetic biology field, since increasing the knowledge about linearity and working boundaries of biological phenomena could lead to a more rational design of artificial systems, also through mathematical models, which, for example, have been used here to study hard-to-predict interactions.

Dataset Information

Construction and characterization of a synthetic Baculovirus-inducible 39K promoter.

Background

Results

Conclusions

Publications

Construction and characterization of a synthetic Baculovirus-inducible 39K promoter.

Similar Datasets

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