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Whole-cell biosensors for detection of heavy metal ions in environmental samples based on metallothionein promoters from Tetrahymena thermophila.

Amaro F, Turkewitz AP, Martín-González A, Gutiérrez JC - Microb Biotechnol (2011)

Bottom Line: In the present paper, we report results with two gene constructs using the Tetrahymena thermophila MTT1 and MTT5 metallothionein promoters linked with the eukaryotic luciferase gene as a reporter.Validation of these whole-cell biosensors was carried out using both artificial and natural samples, including methods for detecting false positives and negatives.Comparison with other published cell biosensors indicates that the Tetrahymena metallothionein promoter-based biosensors appear to be the most sensitive eukaryotic metal biosensors and compare favourably with some prokaryotic biosensors as well.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología-III, Facultad de Biología, C/. José Antonio Novais 2, Universidad Complutense, Madrid, Spain.

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The pMTT1LucFF (6.3 Kb) and pMTT5LucFF (5.3 Kb) plasmid constructs. Electrophoresis analysis shows the expected SacI/KpnI double digest products (4.5 and 3.5 kb in size, arrows) from plasmids pMTT1LucFF (lane A) and pMTT5LucFF (lane B). M1: size marker M1 Kb (Roche). Features of these constructs are indicated in panels A and B (see the text for explanation). BTU2: polyadenylation sequence from BTU2 gene.
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f1: The pMTT1LucFF (6.3 Kb) and pMTT5LucFF (5.3 Kb) plasmid constructs. Electrophoresis analysis shows the expected SacI/KpnI double digest products (4.5 and 3.5 kb in size, arrows) from plasmids pMTT1LucFF (lane A) and pMTT5LucFF (lane B). M1: size marker M1 Kb (Roche). Features of these constructs are indicated in panels A and B (see the text for explanation). BTU2: polyadenylation sequence from BTU2 gene.

Mentions: To create the reporter constructs MTT1::lucFF and MTT5::lucFF, we placed the eukaryotic luciferase gene (lucFF) as a reporter under the transcriptional control of the promoters for the T. thermophila MTT1 or MTT5 genes, as described in Methods. These constructs were each integrated and driven to fixation at the non‐essential btu1‐1 locus in the T. thermophila macronucleus to create stable cell lines. The resulting plasmids pMTT1Luc and pMTT5Luc were restriction digested with KpnI and SacI to release the reporter constructs (MTT1::LucFF or MTT5::LucFF) (Fig. 1), which were then introduced into T. thermophila CU522 strain by biolistic bombardment (Cassidy‐Hanley et al., 1997). The linearized constructs were designed to undergo homologous recombination at the BTU1 locus, which in the CU522 strain bears a paclitaxel‐hypersensitive allele, btu‐1–1 (Gaertig et al., 1994). Consequently, the desired recombinants could be selected based on their paclitaxel resistance. These cell lines were named MTT1Luc and MTT5Luc (Table 1). Starting with these paclitaxel‐resistant isolated clones, we then obtained strains with complete macronuclear replacement (i.e. homozygous strains for btu‐1–1::(MTT1 or MTT5)lucFF by taking advantage of phenotypic assortment (Gaertig et al., 1994).


Whole-cell biosensors for detection of heavy metal ions in environmental samples based on metallothionein promoters from Tetrahymena thermophila.

Amaro F, Turkewitz AP, Martín-González A, Gutiérrez JC - Microb Biotechnol (2011)

The pMTT1LucFF (6.3 Kb) and pMTT5LucFF (5.3 Kb) plasmid constructs. Electrophoresis analysis shows the expected SacI/KpnI double digest products (4.5 and 3.5 kb in size, arrows) from plasmids pMTT1LucFF (lane A) and pMTT5LucFF (lane B). M1: size marker M1 Kb (Roche). Features of these constructs are indicated in panels A and B (see the text for explanation). BTU2: polyadenylation sequence from BTU2 gene.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815263&req=5

f1: The pMTT1LucFF (6.3 Kb) and pMTT5LucFF (5.3 Kb) plasmid constructs. Electrophoresis analysis shows the expected SacI/KpnI double digest products (4.5 and 3.5 kb in size, arrows) from plasmids pMTT1LucFF (lane A) and pMTT5LucFF (lane B). M1: size marker M1 Kb (Roche). Features of these constructs are indicated in panels A and B (see the text for explanation). BTU2: polyadenylation sequence from BTU2 gene.
Mentions: To create the reporter constructs MTT1::lucFF and MTT5::lucFF, we placed the eukaryotic luciferase gene (lucFF) as a reporter under the transcriptional control of the promoters for the T. thermophila MTT1 or MTT5 genes, as described in Methods. These constructs were each integrated and driven to fixation at the non‐essential btu1‐1 locus in the T. thermophila macronucleus to create stable cell lines. The resulting plasmids pMTT1Luc and pMTT5Luc were restriction digested with KpnI and SacI to release the reporter constructs (MTT1::LucFF or MTT5::LucFF) (Fig. 1), which were then introduced into T. thermophila CU522 strain by biolistic bombardment (Cassidy‐Hanley et al., 1997). The linearized constructs were designed to undergo homologous recombination at the BTU1 locus, which in the CU522 strain bears a paclitaxel‐hypersensitive allele, btu‐1–1 (Gaertig et al., 1994). Consequently, the desired recombinants could be selected based on their paclitaxel resistance. These cell lines were named MTT1Luc and MTT5Luc (Table 1). Starting with these paclitaxel‐resistant isolated clones, we then obtained strains with complete macronuclear replacement (i.e. homozygous strains for btu‐1–1::(MTT1 or MTT5)lucFF by taking advantage of phenotypic assortment (Gaertig et al., 1994).

Bottom Line: In the present paper, we report results with two gene constructs using the Tetrahymena thermophila MTT1 and MTT5 metallothionein promoters linked with the eukaryotic luciferase gene as a reporter.Validation of these whole-cell biosensors was carried out using both artificial and natural samples, including methods for detecting false positives and negatives.Comparison with other published cell biosensors indicates that the Tetrahymena metallothionein promoter-based biosensors appear to be the most sensitive eukaryotic metal biosensors and compare favourably with some prokaryotic biosensors as well.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología-III, Facultad de Biología, C/. José Antonio Novais 2, Universidad Complutense, Madrid, Spain.

Show MeSH
Related in: MedlinePlus