# Presentation: 184922

## 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

## MX

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

**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

**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 **

**Test organisms: *****P. putida***** strains**

## 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**

***P. putida***

** ****Corvallis**

- 1

- 10

- 100

- 1000

- 10000

- 100000

- 1000000

- 10000000

- 100000000

- 0.001

- 0.01

- 0.1

- 1

- 10

- 100

**mg/L of Cd****2+**

**# cells per 1 ml**

## Generation of Luciferase Biosensor

**Wild type**

**Light**

**Metal stimulus**

**promoter**

***lux A/B***

**ORF**

**Protein product**

**Transcript **

**Transcript**

**Normal cell**

**Biosensor**

## Slide 8

## Cadmium detection

**	****sensitive**
**Ca inhibits the Cd response**

_**Cadmium detection**_

***P. putida***

** ****Corvallis mutants-Cd **

**[in .01M Ca(NO****3****)****2****]**

- 0

- 2

- 4

- 6

- 8

- 10

- 12

- 14

- 0

- 1

- 10

- 50

- 100

**mg/L Cd****2+**

**Lux**

- Cat A*10

- Bfr

- FeSOD

## Response of KT2440 mutants to Cu

**Response of KT2440 mutants to Cu**

- 0

- 0.5

- 1

- 1.5

- 2

- 2.5

- 3

- 3.5

- 68

- 18

- 45

- 47

- 41

- 4

- 43

- 66

- 70

- 69

- 7

- 31

- 46

- 21

- 3

- 30

**Mutant strain**

**Relative change in lux (fold)**

- 10 mg/L

- 1 mg/L

## Response of KT2440 mutants to Cd

**Response of KT2440 mutants to Cd**

- 0

- 2

- 4

- 6

- 8

- 10

- 12

- 14

- 16

- 54

- 18

- 31

- 30

- 43

- 4

- 68

- 46

- 45

- 69

- 7

- 70

- 21

- 41

- 3

- 47

- 45

**Mutant strain**

**Relative change in lux (fold)**

- 100 mg/L

- 10 mg/L

- 1 mg/L

## pI 4			pI 7

- pI 4			pI 7

- pI 4			pI7

## Spot B: increased three fold

**Spot B: increased three fold**
**Corresponded to lipoamide dehydrogenase in TCA cycle**

## pI 7

**Mr (kDa)**

**97.0**

**66.0**

**45.0**

**30.0**

**20.1**

**14.4**

**KT2440    0.01M Ca(NO****3****)****2 ****    ****100 ppm Cd**

**4-15%**

**pI 4**

**pI 7**

- Peptides of increased
- Intensity.

## Conclusions

**RD-83090701-0**

**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**