Results.html



 

RESULTS:

1.  Promoter Characterization:

 

Promoter consists of several discrete domains arranged in a specific orientation (modular in nature), such that redesigning of the ‘cis-architecture’ of a promoter DNA across its sequence backbone is feasible (Gurr and Rushton 2005; Rushton et al. 2002).

The resultant functionality of the newly derived synthetic module depends upon the altered positioning of discrete cis motif/s with respect to their positions, spacing, orientation and copy number/s (Fessele et al. 2002; Tjian and Maniatis 1994).

In an earlier study conducted by our primary investigator a very high expression was observed in Mirabalis Mosaic Virus promoter (pPMMVFLt12), thereby for the present study the strong promoter having the synthetic cis elements upstream was designed using online based software PLANT CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Figure 1: represents a pictorial view of cis-elements present in pPMMVFLt12 promoter.

Fig 1: Pictorial view of cis-elements present in pPMMVFLt12 promoter.

2.  Cloning of Metallothionein:

Two different parts were created for our project:

1.      BBa_K2441000: MT4MBP (Top-4 metal binder protein; Modified iGEM part BBa­_K1478002), this part codes for a metal binding protein capable of binding Zinc (Zn), Cadmium (Cd), Copper (Cu), and Arsenic (As).

2.      Human Metal Binding Protein-3 this part codes for a metal binding protein capable of binding Zinc, Cadmium, Copper, and Arsenic.

#unfortunately we are unable to submit this part due to its bio-brick incompatibility.

Successful clones are obtained (Figure 2). Sequencing was done in duplicates (two colonies of each clone) for sequence validation.

Figure 2:  Picture of Agarose Gel conducted during cloning Experiments.

 

 

3.  Bio-Beads Formation:

Different Concentration of Calcium Chloride was tested with varied concentration of Na-Alginate, among which beads formed in 1% Na-Alginate in 100 mM Cacl2 had shown the best results in terms of their shape and expressivity.

Bio-Beads were made from different cells of E. Coli BL-21 expressing our gene of Interest i.e MT4MBP and HMP of promoter our synthetically characterized promoter (Figure 4).

 

Fig 3: Immobilization of E Coli BL-21 cells expressing our protein of Interest

(a)    Only Bio-Beads without any bacterial cells

(b)   E.coli Bio-Beads does not expressing our protein of Interest

(c)    E. coli Bio-Beads expressing MT4MBP

(d)   E. coli Bio-Beads expressing HMP3

 

 

 

 

 

 

 

 

Figure 4: Treatment of water samples with form Bio-Beads

(a)    Treatment of artificial water having 0.5mM CuCl2, 0.5mM ZnCl2 and 0.5mM CdCl2.  with formed Bio-Beads

(b)   Treatment of Sewage Treatment Plant  Inlet Water  with formed Bio-Beads

(c)    Treatment of Sewage Treatment Plant  Outlet Water  with formed Bio-Beads
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 



Figure 5: Visual observation of beads turning blue in 0.05 mM CuCl2 Solution after absorption with heavy metals

 

 

 

 

5.  ICP OES: Heavy Metal Detection

Artificial water was created in laboratory having 0.5 mM (milli molar) of Calcium Chloride, Zinc Chloride, Cadmium Chloride was treated with Bio-Beads made up of cells expressing our gene of interest with two different sets of promoters. The heavy metal concentration was tested via ICP OES with a dilution factor of 50 (Table 1).

v  Arsenic was not tested due to Biosafety issues.

Table 1: Detection of heavy metals in water sample via ICP OES

Heavy Metal

S. No

Bio-Beads

Artificial Water

Test Sample 1

Test Sample 2

 

 

 

Reading

(mg/L)

Percentage Decrease

(with respect to positive control)

Reading

(mg/L)

Percentage Decrease

 

Reading

(mg/L)

Percentage Decrease

 

Copper (Cu)

1

Negative Control

-0.1350

-NA-

-0.1781

-NA-

-0.1871

-NA-

2

Positive Control

2.19540

0.00

5.7865

0.00

5.1789

0.00

3

MMV-MT4MBP

1.07865

50.87

3.892

32.73

3.701

28.53

4

MMV-HMP

0.36295

83.46

3.167

45.269

2.987

42.32

Zinc (Zn)

1

Negative Control

-0.1562

-NA-

-0.1348

-NA-

-0.1671

-NA-

2

Positive Control

51.09600

0.00

6.04695

0.00

5.9879

0.00

3

MMV-MT4MBP

40.01135

21.63

5.0892

15.80

5.1981

13.18

4

MMV-HMP

35.24370

31.02

4.9017

18.9

5.6897

4.98

Cadmium (Cd)

1

Negative Control

-0.1567

-NA-

-0.1678

-NA-

-0.1891

-NA-

2

Positive Control

73.4245

0.00

8.4955

0.00

7.9676

0.00

3

MMV-MT4MBP

72.2775

1.56

8.071

4.99

6.6714

16.27

4

MMV-HMP

54.3715

25.94

7.8991

7.02

6.2019

22.16

 

 

 

Figure 6(a): Linearity curve for Cd, Cu and Zn   

 

Figure 6 (b): Peaks obtained of heavy metals for corresponding samples

 

 

 

 

 

 

 

Conclusion

Team DEI AGRA had initiated the project entitled as “Bio-Beads for removing heavy metal toxicity from Industrial effluents”, with a vision of contributing to a solution for a world-wide problem of heavy metal toxicity, which possess a serious threat to human health.

We had accomplished our objectives of cloning the mettalloprotein downstream to our characterized synthetic promoter, which are successfully immobilized in forms of Bio Beads. Our results indicated more than 80% decrease of copper, while 30% and 20% reduction in Zinc (Zn) and Cadmium (Cd) respectively in case of artificial water having 0.5mM CuCl2, 0.5mM ZnCl2 and 0.5mM CdCl2, while lesser but significant decrement observed in test samples collected from Sewage Treatment Plant of Agra, India.

 

 

 

 

Inference

We had fabricated Bio-Beads by immobilizing bacterial cells, which strongly expressing MT4MBP and HMP3 for fast, easy and user handy purification of water possessing heavy metal toxicity.