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Biosensors: Development and Environmental Testing Investigators: Anne J. Anderson, Charles D. Miller and Joan E. McLean Utah State University Logan, Utah 84322-5305. RD-83090701-0. Metals of interest Copper: maximum drinking water level standard 1.3 mg/L

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Biosensors: Development and Environmental Testing

Investigators:

Anne J. Anderson, Charles D. Miller and Joan E. McLean

Utah State University

Logan, Utah 84322-5305

RD-83090701-0


Metals of interest

Copper:maximum drinking water level standard 1.3 mg/L

aquatic chronic exposure limit 30.3 µg/L

Human tolerance relatively high

Used for microbial pest control

Some lower organisms acute toxicity (phytoplankton, zooplankton,

amphibians)

Cadmium:maximum drinking water standard 0.005 mg/L

aquatic chronic exposure limit 2.7µg/L

Higher animal toxicity: accumulates in kidneys and liver,

produces bone and blood problems

Carcinogenic (Group B1)

Minor use as pesticide


Cu is a trace element: required for certain enzymatic and carrier proteins

Cd no known cellular use—toxic- may disrupt Ca and Zn metabolism

Both cause oxidative stress

Both act on –SH proteins

Cell concentrations regulated by chelation, sorption, uptake, and efflux mechanisms

MX


  • Biosensors

  • Final goals:

  • An array of promoter fusions that respond differentially and specifically

  • with light output upon exposure to toxic metals

  • 2) A gene chip array to detect transcript abundance from cells responding to

  • toxic metals. Pattern of gene activation would specify the metal.


Test organisms: P. putida strains

Cells of both pseudomonads are killed by concentrations of Cu

starting between 1 and 10 mg/L


P. putida KT2440

1000000000

100000000

10000000

1000000

100000

1 h

1 h

# cells per 1 ml

4 h

4 h

10000

1000

100

10

1

0.001

0.01

0.1

1

10

100

mg/L of Cd2+

P. putida

Corvallis

100000000

10000000

1000000

100000

10000

# cells per 1 ml

1000

100

10

1

0.001

0.01

0.1

1

10

100

mg/L of Cd2+

The pseudomonad cells show little change in culturability with Cd

even at doses of 100 mg/L.

Both wild type isolates behave similarly showing resistance to Cd


Generation of Luciferase Biosensor

Metal stimulus

Wild type

lux A/B

promoter

ORF

Transcript

Transcript

Light

Protein product

Normal cell

Biosensor


P. putida

Corvallis mutants-Cu [in .01M CaCl2]

30

25

20

Cat A*10

Lux

Bfr

15

FeSOD

10

5

0

0

0.01

0.1

1

10

mg/L of Cu2+


P. putida

Corvallis mutants-Cd [in .01M

KNO

]

3

14

12

10

Cat A*10

8

Lux

Bfr

6

FeSOD

4

2

0

0

1

10

50

100

mg/L of Cd2+

Corvallis mutants-Cd

P. putida

Cadmium detection

[in .01M Ca(NO3)2]

14

Cat A*10

12

Bfr

10

FeSOD

8

Lux

6

4

2

0

0

1

10

50

100

mg/L Cd2+

FeSOD mutant is most

sensitive

Ca inhibits the Cd response


Response of KT2440 mutants to Cu

3.5

3

2.5

2

10 mg/L

Relative change in lux (fold)

1 mg/L

1.5

1

0.5

0

68

18

45

47

41

4

43

66

70

69

7

31

46

21

3

30

Mutant strain


Response of KT2440 mutants to Cd

16

14

12

10

100 mg/L

10 mg/L

8

Relative change in lux (fold)

1 mg/L

6

4

2

0

54

18

31

30

43

4

68

46

45

69

7

70

21

41

3

47

45

Mutant strain


pI 4 pI 7

pI 4 pI7


Spot B: increased three fold

Corresponded to lipoamide dehydrogenase in TCA cycle


pI 4

pI 7

4-15%

Mr (kDa)

Cd responses

97.0

66.0

45.0

30.0

20.1

14.4

Peptides of increased

Intensity.

KT2440 0.01M Ca(NO3)2 100 ppm Cd


Conclusions

  • The two approaches for biosensor development are feasible

  • 2) luxAB::Insertional mutants that detect Cu and Cd differentially have

  • been identified

  • Gene loci await determination

  • 3) Peptides that increase upon Cu exposure are detected and one has been

  • correlated with a specific function

RD-83090701-0


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