# US 8,785,174 B2 · Heavy metal remediation system

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- Number: US 8,785,174 B2
- Document ID: US8785174B2
- Status: Granted
- Kind: B2
- Application number: 12/859,590
- Filed: 2010-08-19
- Published: 2011-09-15
- Granted: 2014-07-22
- Earliest claimed: 2009-08-20
- Assignees: Inter American University of Puerto Rico
- Applicants: Oscar N. Ruiz
- Inventors: Oscar N. Ruiz
- Web page: https://patentgrep.com/patent/US8785174B2

## CPC

- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C02F 3/341: {Consortia of bacteria}
- C12N 1/20: Bacteria; Culture media therefor
- C12Q 1/34: involving hydrolase
- G01N 33/523: {the element being adapted for a specific analyte}
- G01N 33/84: involving inorganic compounds or pH
- C02F 2101/20: Heavy metals or heavy metal compounds
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C02F: TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE (separation in general B01D; special arrangements on waterborne vessels of installations for treating water, waste water or sewage, e.g. for producing fresh water, B63J; adding materials to water to prevent corrosion C23F; treating radioactively-contaminated liquids G21F9/04; regeneration of reactants for recirculation into processes, see the relevant places for the processes)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention provides a system of heavy metal sequestration by bacteria. The bacteria expresses the ppk, mt, and/or β-galactosidase (lacZ) genes and can tolerate at least 25 μM mercury, 1,000 μM zinc, 250 μM cadmium, and 3,000 μM Pb. The system allows for facile determination of the presence of heavy metal contaminants in a liquid and the facile collection of the bacteria that has sequestered large amounts of heavy metal. Further provided is a system of gene expression in bacteria that comprises phage and plastid gene expression elements and delivers a particularly high level of protein expression and heavy metal resistance.

## Claims

### Claim 1

A bacterium comprising a transgenic non-fusion mt1 chelator agent, wherein the bacterium is resistant to between at least 20 μM and about 160 μM Hg.

### Claim 2 (depends on 1)

The bacterium of claim 1, which is resistant to between at least 20 gM and about 140 pM Hg.

### Claim 3 (depends on 2)

The bacterium of claim 2, which is resistant to between at least 38 μM and about 140 μM Hg.

### Claim 4 (depends on 2)

The bacterium of claim 2, which bacterium is not transgenic in respect to a mer operon functions affecting heavy metal transport to the cytoplasm.

### Claim 5 (depends on 2)

The bacterium of claim 2, wherein the chelator agent gene is transcribed from a strong promoter and is flanked by a 5′ UTR and a 3′ UTR, whereby at least between 4,000 and 8,500 copies of stable transcripts per ng total mRNA correspond to the chelator gene.

### Claim 6 (depends on 5)

The bacterium of claim 5, wherein at least between 6,000 and 7,500 copies of stable transcripts per ng total mRNA correspond to the chelator gene.

### Claim 7 (depends on 5)

The bacterium of claim 5, wherein the chelator agent gene is flanked by a 5′ UTR translational enhancer element sequence derived from bacteriophage T7 gene 10 and a plastid rps16 gene 3′UTR Rho-independent transcriptional terminator sequence.

### Claim 8 (depends on 5)

The bacterium of claim 5, wherein the chelator agent gene is transcribed from a transcriptional constitutive promoter sequence derived from the plastid 16S rrn gene and is flanked by a 5′ UTR transcriptional enhancer element sequence derived from bacteriophage T7 gene 10 and a plastid rps16 gene 3′UTR Rho-independent transcriptional terminator sequence.

### Claim 9 (depends on 5)

The bacterium of claim 5, where the bacterium is resistant to above 20 μM up to about 140 μM mercury and cadmium at concentrations above about 200 μM up to about 250 μM, zinc at concentrations above about 500 μM up to about 1000 μM, or lead at concentrations above about 250 μM up to about 3,000 μM.

### Claim 10 (depends on 2)

The bacterium of claim 2, which, when in a liquid environment containing mercury, accumulates the mercury and turns dark in coloring.

### Claim 11 (depends on 2)

The bacterium of claim 2, which, when in a liquid environment containing mercury, cadmium, zinc or lead, accumulates the mercury, cadmium, zinc or lead and turns dark in coloring.

### Claim 12 (depends on 1)

The bacterium of claim 1, selected from among E. coli, Pseudomonas, Cyanobacteria and Bacillus.

### Claim 13 (depends on 1)

The bacterium of claim 1, which, when in a liquid environment containing mercury, cadmium, zinc or lead, it accumulates the mercury, cadmium, zinc or lead, forms aggregates and precipitates.

### Claim 14 (depends on 1)

The bacterium of claim 1, grown in a biofilter for convenient handling to remove heavy metal from a contaminated liquid.

### Claim 15 (depends on 1)

The bacterium of claim 1, wherein the chelator agent is not a fusion protein.

### Claim 16 (depends on 4)

The bacterium of claim 4, wherein the chelator agent is not a fusion protein.

### Claim 17 (depends on 1)

The bacterium of claim 1, wherein the chelator agent gene is transcribed from a strong promoter and is flanked by a 5′ UTR and a 3′ UTR, whereby at least between 4,000 and 8,500 copies of stable transcripts per ng total mRNA correspond to the chelator gene, and whereby the bacterium is resistant to between at least 20 μM and about 160 μM Hg.

### Claim 18

A bacterium comprising a pBSK-P16S-g10-lacZ-3′UTR plasmid wherein the ability of β-galactosidase to cleave 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) is reduced by the presence of mercury.