Reporter gene (Luciferase) assay

Introduction：

Reporter genes provide a convenient, simple and rapid way for assay of gene expression regulation. Commonly used reporter genes include: b-galactosidase, green fluorescence protein, firefly luciferase and bacterial luciferase. The bacterial luciferase utilizes long chain aldehydes as a substrate to produce light. The instrument measuring the bioluminescence is called a luminometer. The intensity of the light reflects the expression level.

Purpose：

Using the bacterial luciferase as a reporter to investigate gene regulation. We design procedure to investigate the effects of IPTG/ lactose/ glucose on the lac promoter, and then measure the intensity of light at different time course.  

Materials：

  E. coli XL-1Blue + pUCD1752 (a plasmid containing the luxAB genes under the control of lac promoter)

  E. coli BL-21 + pHPA129,pHPA31 (a plasmid containing the luxAB genes under the control of acetoin dehydrogenase promoter)

   E. coli XL-1Blue (negative control)

  LB-ampicillin broth (A-100, Cm30Kan25)

  20mM Glucose/0.1mM lactose/0.1mM IPTG/0.01% acetoin

   0. 1% n-decyl aldehyde in 2mg/ml bovine serum albumin (BSA)

   Luminometer, test tube, spectrophotometer, cuvette

Mathods：

I

5x dilute in LB-A100 broth

IPTG

Inoculation time

1

E. coli XL-1Blue + pUCD1752

1mM

30min

2

E. coli XL-1Blue + pUCD1752

1mM

1hr

3

E. coli XL-1Blue + pUCD1752

1mM

1hr & 30min

4

E. coli XL-1Blue + pUCD1752

1mM

2hr

5

E. coli XL-1Blue (dilute in LB)

1mM

2hr

II

5x dilute in LB-A100 broth

Glucose

Inoculation time

1

E. coli XL-1Blue + pUCD1752

20mM

30min

2

E. coli XL-1Blue + pUCD1752

20mM

1hr

3

E. coli XL-1Blue + pUCD1752

20mM

1hr & 30min

4

E. coli XL-1Blue + pUCD1752

20mM

2hr

5

E. coli XL-1Blue (dilute in LB)

20mM

2hr

 

III

5x dilute in LB-A100 broth

Lactose

Inoculation time

1

E. coli XL-1Blue + pUCD1752

1mM

30min

2

E. coli XL-1Blue + pUCD1752

1mM

1hr

3

E. coli XL-1Blue + pUCD1752

1mM

1hr & 30min

4

E. coli XL-1Blue + pUCD1752

1mM

2hr

5

E. coli XL-1Blue (dilute in LB)

1mM

2hr

 

↓

Mix 500 μl bacteria and 500μl 0.1% n-decyl aldehyde in 2 mg/ml bovine serum albumin (BSA). Measure the luminescence in a Luminometer.

 

Results：  

A)

30min

I-1

II-1

III-1

OD600

0.199

Sample lost

0.322

Rdy

231.8

X

71.44

RLU

1164.8

X

221.9

 

1hr

I-2

II-2

III-2

OD600

0.240

0.289

0.277

Rdy

468.7

341.1

185.3

RLU

1952.9

1180.3

669.0

 

1hr & 30min

I-3

II-3

III-3

OD600

0.457

0.371

0.443

Rdy

278.3

280

143.7

RLU

609.0

754.7

324.4

 

2hr

I-4

II-4

III-4

I-5

II-5

III-5

OD600

0.534

0.540

0.555

0.340

0.264

0.381

Rdy

518.6

447.5

144.0

0.111

0.104

0.136

RLU

971.2

828.7

259.5

0.326

0.394

0.357

 

 

B) Inoculate 1hr →靜置一小時後再測OD及Rdy

1hr

I-2

II-2

III-2

OD600

0.090

0.121

0.059

Rdy

1077

829.4

387.2

RLU

11966.7

6854.5

6562.7

 

Discussion：