Dataset Information

Identification of an intestine-specific promoter and inducible expression of bacterial ?-galactosidase in mammalian cells by a lac operon system.

ABSTRACT: UNLABELLED: BACKGROUND:?-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors (such as ?-galactoside) in feed. Intestine-specific and substrate inducible expression of ?-galactosidase would be highly beneficial for transgenic animal production. METHODS:To achieve the intestine-specific and substrate inducible expression of ?-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tissue specificity of four intestine-specific promoters from human intestinal fatty acid binding protein, rat intestinal fatty acid binding protein, human mucin-2 and human lysozyme. We made two chimeric constructs combining the promoter and enhancer of human mucin-2, rat intestinal trefoil factor and human sucrase-isomaltase. Then a modified lac operon system was constructed to investigate the induction of ?-galactosidase expression and enzyme activity by isopropyl ?-D-1-thiogalactopyranoside (IPTG) and an ?-galactosidase substrate, ?-lactose.We declared that the research carried out on human (Zhai Yafeng) was in compliance with the Helsinki Declaration, and experimental research on animals also followed internationally recognized guidelines. RESULTS:The activity of the human mucin-2 promoter was about 2 to 3 times higher than that of other intestine-specific promoters. In the lac operon system, the repressor significantly decreased (P < 0.05) luciferase activity by approximately 6.5-fold and reduced the percentage of cells expressing green fluorescent protein (GFP) by approximately 2-fold. In addition, the expression level of ?-galactosidase mRNA was decreased by 6-fold and ?-galactosidase activity was reduced by 8-fold. In line with our expectations, IPTG and ?-lactose supplementation reversed (P < 0.05) the inhibition and produced a 5-fold increase of luciferase activity, an 11-fold enhancement in the percentage of cells with GFP expression and an increase in ?-galactosidase mRNA abundance (by about 5-fold) and ?-galactosidase activity (by about 7-fold). CONCLUSIONS:We have successfully constructed a high specificity inducible lac operon system in an intestine-derived cell line, which could be of great value for gene therapy applications and transgenic animal production.

SUBMITTER: Ya-Feng Z

PROVIDER: S-EPMC3527164 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Identification of an intestine-specific promoter and inducible expression of bacterial α-galactosidase in mammalian cells by a lac operon system.

Ya-Feng Zhai Z Gang Shu S Xiao-Tong Zhu Z Zhi-Qi Zhang Z Xia-Jing Lin L Song-Bo Wang W Li-Na Wang W Yong-Liang Zhang Z Qing-Yan Jiang J

Journal of animal science and biotechnology 20121030 1

<h4>Unlabelled</h4><h4>Background</h4>α-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors (such as α-galactoside) in feed. Intestine-specific and substrate inducible expression of α-galactosidase would be highly beneficial for transgenic animal production.<h4>Methods</h4>To achieve the intestine-specific and substrate inducible expression of α-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tiss ...[more]

PMID: 23111091

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Project description:1. Several lac diploid strains of Escherichia coli were constructed and tested to discover whether mutations in the lac promoter alleviate catabolite repression. 2. In each of these diploids the chromosome carries one of the promoter mutations, L8, L29 or L1; so that the rate of synthesis of the enzymes of the lac operon is only 2-6% of the fully induced wild-type. Each diploid harbours the episome F'lacM15 that specifies the synthesis of thiogalactoside transacetylase under the control of intact regulator, promoter and operator regions, but has a deletion in the structural gene for beta-galactosidase. In each diploid more than 90% of the thiogalactoside transacetylase is synthesized from the episome, and 100% of the beta-galactosidase is synthesized from the chromosome, and comparison of the extent of catabolite repression that the two enzymes suffered indicated whether the chromosomal promoter mutation relieves catabolite repression. 3. In the strains in which the promoter carries either of the point mutations L8 or L29 the enzymes were equally repressed, suggesting that neither L8 nor L29 affects catabolite repression. 4. In a diploid strain harbouring the same episome but carrying deletion L1 on the chromosome, synthesis of beta-galactosidase suffered much less repression than that of thiogalactoside transacetylase. 5. In a diploid strain in which the chromosome carries L1 and also a second mutation that increases the rate of expression of lac to that permitted by L8 or L29, the synthesis of beta-galactosidase again suffered much less repression than the synthesis of thiogalactoside transacetylase. 6. The effect of L1 (which deletes the boundary between the i gene and the lac promoter) is ascribed to its bringing the expression of lac under the control of the promoter of the i gene. 7. Even in strains carrying L1, some catabolite repression persists; this is not due to a trans effect from the episome since it occurs equally in a haploid strain with L1.

Project description:The role of stochasticity on gene expression is widely discussed. Both potential advantages and disadvantages have been revealed. In some systems, noise in gene expression has been quantified, in among others the lac operon of Escherichia coli. Whether stochastic gene expression in this system is detrimental or beneficial for the cells is, however, still unclear. We are interested in the effects of stochasticity from an evolutionary point of view. We study this question in the lac operon, taking a computational approach: using a detailed, quantitative, spatial model, we evolve through a mutation-selection process the shape of the promoter function and therewith the effective amount of stochasticity. We find that noise values for lactose, the natural inducer, are much lower than for artificial, nonmetabolizable inducers, because these artificial inducers experience a stronger positive feedback. In the evolved promoter functions, noise due to stochasticity in gene expression, when induced by lactose, only plays a very minor role in short-term physiological adaptation, because other sources of population heterogeneity dominate. Finally, promoter functions evolved in the stochastic model evolve to higher repressed transcription rates than those evolved in a deterministic version of the model. This causes these promoter functions to experience less stochasticity in gene expression. We show that a high repression rate and hence high stochasticity increases the delay in lactose uptake in a variable environment. We conclude that the lac operon evolved such that the impact of stochastic gene expression is minor in its natural environment, but happens to respond with much stronger stochasticity when confronted with artificial inducers. In this particular system, we have shown that stochasticity is detrimental. Moreover, we demonstrate that in silico evolution in a quantitative model, by mutating the parameters of interest, is a promising way to unravel the functional properties of biological systems.

Dataset Information

Identification of an intestine-specific promoter and inducible expression of bacterial ?-galactosidase in mammalian cells by a lac operon system.

Publications

Identification of an intestine-specific promoter and inducible expression of bacterial α-galactosidase in mammalian cells by a lac operon system.

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