LABORATORY ORIENTED PROJECT

A report on:

Biosynthesis of Tellurium

Nanoparticles and Protein Profile

Studies on Marine Bacteria

Submitted to

Dr. Meenal KowshikAssociate Professor

BITS Pilani K K Birla Goa Campus

By

Edarapalli V R Nikhil

2011B1A1673G

Contents A report on: Biosynthesis of Tellurium Nanoparticles and Protein Profile Studies on Marine

Bacterium ............................................................................................................................................... 1

Submitted to Dr. Meenal Kowshik Associate Professor BITS Pilani K K Birla Goa Campus ... 1

Introduction: ........................................................................................................................................... 3

Tellurite (K2TeO3) Concentration ............................................................................................................ 4

Biosynthesis and Dialysis ........................................................................................................................ 4

SDS-PAGE- Standardization ..................................................................................................................... 6

Protocol: .......................................................................................................................................... 6

SDS PAGE Chemicals ....................................................................................................................... 8

Standardization: .............................................................................................................................. 8

Experiments: ................................................................................................................................. 10

Lowry’s method: ........................................................................................................................... 13

Results & Discussions: ........................................................................................................................... 15

Lowry’s Method: ........................................................................................................................... 15

SDS PAGE gels: .............................................................................................................................. 15

Conclusion: ............................................................................................................................................ 16

Protein Profiles ...................................................................................................................... 16

The Standard Graph For Lowry’s Method:............................................................................ 16

Bibliography: ......................................................................................................................................... 17

Introduction: Microbial resistance to tellurite is a widespread phenomenon. In most

environments, tellurite resistant organisms comprise upto 10% of total culturable microbial

population. The biological significance of this relatively common trait is not yet known

though resistance to oxidative stress and reactive oxygen species has been proposed based

on the studies done on Escherichia coli. E coli is sensitive to tellurite relative to many other

tellurite resistance isolates.

Microbial resistance to most toxic heavy metals is due to their chemical

detoxification as well as due to energy dependent ion efflux from the cell by membrane

proteins that function either as ATPase or as chemiosmotic cation or proton anti

transporters. Therefore microbial systems can detoxify the metal ions by either reduction

and/or precipitation of soluble toxic inorganic ions to insoluble non-toxic metal nano-

clusters.

Some studies report microbial strains isolated aerobically on the basis of tellurite

resistance and subsequently examined for their capacity to volatilize tellurium in pure

cultures. The tellurite-resistant strains recovered were either yeasts related to marine

isolates of Rhodotorula spp. or gram-positive bacteria related to marine strains within the

family Bacillaceae based on rRNA gene sequence comparisons. Most strains produced

volatile tellurides, primarily dimethyltelluride, though there was a wide range of the types

and amounts of species produced

In this Project, the protein profiling of idiomarina loihiensis (here after: ML2, “Mine

Loading area-2 sample”) is done to check effects of age on the volatilization of tellurium

nanoparticles which were synthesized by the bacteria itself.

Tellurite (K2TeO3) Concentration The concentration at which the growth and synthesis of ML2 need to found. 5

samples were subjected to different concentration of tellurite salt and incubated at 370C for

40 hr, and their growth is observed.

Te (mM)

Inoculum (μL)

Tellurite (μL)

0.05 200 10

0.1 200 20

0.25 200 50

0.5 200 100

0.1 200 200

Cntrl 0 200

It is observed that the culture at 0.25mM concentration of Tellurite salt showed

better growth. Hence, this concentration was applied in experiments here after.

Biosynthesis and Dialysis 500 mL of Zobell Marine Broth was inoculated with ML2 (inoculum volume=5%)

along with tellurite salt of concentration 0.25 mM, and incubated in shaker maintained at

370C for 40-48 hours. And later centrifuged and discarding the supernatant, rest is carried

forward for Dialysis

Dialysis is the process of separating molecules in solution by the difference in their

rates of diffusion through a semipermeable membrane, such as dialysis tubing in these

experiments. Due to the pore size of the membrane, large molecules in the sample cannot

pass through the membrane, thereby restricting their diffusion from the sample chamber.

By contrast, small molecules will freely diffuse across the membrane and obtain equilibrium

across the entire solution volume, thereby changing the overall concentration of these

molecules in the sample and dialysate. Once equilibrium is reached, the final concentration

of molecules is dependent on the volumes of the solutions involved, and if the equilibrated

dialysate is replaced with fresh dialysate, diffusion will further reduce the concentration of

the small molecules in the sample.

The rate of dialysis is also directly proportional to the surface area of the membrane

and inversely proportional to its thickness. Membranes normally used for laboratory dialysis

applications are 0.5 to 1.2 mil (12 to 30µm) thick, providing good diffusion rate as well as

structural integrity. While membrane thickness is not a variable that is easily modified, the

surface area usually is. The flatter a sample can be spread over a membrane surface, the

faster will be its dialysis because all molecules in the sample will be closer to the membrane

and a higher proportion of them will be in direct contact with the membrane at any instant.

High-performance dialysis products, such as Thermo-Scientific Slide-A-Lyzer Dialysis

Cassettes, MINI Devices and Flasks, are designed to maximize surface area-to-volume ratios

(within practical limits) for different volumes of sample.

To remove additional unwanted substance, it is necessary to replace the dialysis

buffer so that a new concentration gradient can be established. Once the buffer is changed,

movement of particles from high (inside the membrane) to low (outside the membrane)

concentration will resume until equilibrium is once again reached. With each change of

dialysis buffer, substances inside the membrane are further purified by a factor equal to the

volume difference of the two compartments. For example, if one is dialyzing 1 ml of sample

against 200 ml of dialysis buffer, the concentration of the dialyzable substance at

equilibrium will be diluted 200 less than at the start. Each new exchange against 200 ml of

new dialysis buffer will dilute the sample 200 times more. For example, for three exchanges

of 200 ml, the sample will be diluted 200 x 200 x 200 or 8,000,000 times, assuming complete

equilibrium was reached each time before the dialysis buffer was changed.

The culture was centrifuged at an RPM of 12000 for 15 min, and the supernatant was

discarded. Rest is loaded into dialysis membrane-bags and suspended in beaker with RO

purified water. The water is changed for every 1 hour, for first 3 hours and for every 4 hours

for next 20 hours.

SDS-PAGE- Standardization Sodium dodecyl sulphate polyacrylamide gel electrophores, is the most widely used

technique to separate proteins from complicated samples of mixture, plays key roles in

molecular biology and wide range of subfield of biological research. Being present a

electricity, proteins migrate towards the negative anode inside the poly-acrylamide gel

under denaturing conditions. In SDS-PAGE, the detergent SDS and a heating step determine

that the electrophoretic mobility of a single kind of protein is only affected by its molecular

weight in the porous acrylamide gel.

The SDS PAGE gel in a single electrophoresis run can be divided into stacking gel and

separating gel. Stacking gel is poured on top of the separating gel (after solidification) and a

gel comb is inserted in the stacking gel. The acrylamide percentage in SDS PAGE gel depends

on the size of the target protein in the sample

For example:

Protocol: 1. Make the separating gel:

-Set the casting frames (clamp two glass plates in the casting frames) on the casting stands.

-Prepare the gel solution (as described above) in a separate small beaker.

-Swirl the solution gently but thoroughly.

-Pipet appropriate amount of separating gel solution (listed above) into the gap between

the glass plates.

-To make the top of the separating gel be horizontal, fill in water (either isopropanol) into

the gap until a overflow.

-Wait for 20-30min to let it polymerize.

Make the stacking gel:

-Discard the water and you can see separating gel left.

-Pipet in stacking gel until a overflow.

-Insert the well-forming comb without trapping air under the teeth. Wait for 20-30min to let

it polymerize.

2. Make sure a complete polymerization of the stacking gel and take out the comb. Take the

glass plates out of the casting frame and set them in the cell buffer dam. Pour the running

buffer into the inner chamber and keep pouring after overflow until the buffer surface

reaches the required level in the outer chamber.

3. Prepare the samples:

-Mix your samples with sample buffer (loading buffer).

-Heat them in boiling water for 5-10 min.

4. Load prepared samples into wells and make sure not to overflow. Don't forget loading

protein marker into the first lane. Then cover the top and connect the anodes.

5. Set an appropriate volt and run the electrophoresis when everything's done.

6. As for the total running time, stop SDS-PAGE running when the downmost sign of the

protein marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of

the glass plate. Generally, about 1-2 hour for a 85V voltage and a 12% separating gel. For a

separating gel possessing higher percentage of acylamide, the time will be longer.

SDS PAGE Chemicals

5X Running Buffer:

25 mM Tris 15.1 g/L

250 mM Glycine 94 g/L

0.10% SDS(10%) 50 mL

1.5 M Tris pH 8.8

1 M Tris pH 6.8

10% SDS

Acrylamide mix 30% 29.2 g acrylamide

0.8 g bis acrylamide in 100 mL

CBB Staining Solution

Standardization: The amount of Lysis buffer, loading dye, culture sample etc., for SDS PAGE analysis

were determined by these experiments

The gel was run at following with following compositions of columns

The column #4 showed bands of suitable width and thickness without any widening

which could be due to presence of high salts (observed in other colums). Hence forth, this

particular composition was used.

Experiments:

The ML2 cultures with Tellurite salt of concentration 0.25mM in Zobell Marine Broth

were inoculated at different time, and samples from them are subjected to SDS PAGE

analysis to observe the effect of age on synthesis of TeNPs by ML2

SDS PAGE was done on Day 4

Gel was run with adding any lysis buffer, to check the protein bands. The plot of gel

was as tabulated below.

There bands were observed:

Further, the experiment was repeated with increase in number of controls and different

volumes of flasks but same amount of media in it (changing the ratio of volume to

headspace). Flasks # 9 & 10 are of volume 100 mL, rest all are 50 mL but the media in all is

20 mL.

The gel was run on Day 4

The gel plot:

The bands observed:

To confirm a protein band observed in oldest culture with tellurite salt, the

experiment was repeated again.

The gel with bands was observed to be:

Lowry’s method:

The lowry’s method was done to estimate the protein content in the samples that

were loaded in the SDS PAGE.

The principle behind the Lowry method of determining protein concentrations lies in

the reactivity of the peptide nitrogen with the copper+2 ions under alkaline conditions and

the subsequent reduction of the FolinCiocalteau phosphomolybdic phosphotungstic acid to

hetero-polymolybdenum blue by the copper-catalysed oxidation of aromatic acids. The

Lowry method is sensitive to pH changes and therefore the pH of assay solution should be

maintained at 10 - 10.5. The Lowry method is sensitive to low concentrations of protein. The

major disadvantage of the Lowry method is the narrow pH range within which it is accurate.

However, we will be using very small volumes of sample, which will have little or no effect

on pH of the reaction mixture

Reagents

A. 2% Na2CO3 in 0.1 N NaOH

B. 1% NaK Tartrate in H2O

C. 0.5% CuSO4.5 H2O in H2O

D. Reagent I: 48 ml of A, 1 ml of B, 1 ml C

E. Reagent II- 1 part Folin-Phenol [2 N]: 1 part water

BSA Standards: 100, 150 and 200 μg/mL

Procedure:

0.2 ml of BSA working standard in 5 test tubes and make up to 1ml using distilled water.

• The test tube with 1 ml distilled water serves as blank.

• Add 4.5 ml of Reagent I and incubate for 10 minutes.

• After incubation add 0.5 ml of reagent II and incubate for 30 minutes

• Measure the absorbance at 660 nm and plot the standard graph.

• Estimate the amount of protein present in the given sample from the standard graph

The protein content was found out to be:

Results & Discussions:

Lowry’s Method:

As the cells were lysed, there was SDS present in the samples. SDS interacts with

absorbance. To avoid this, the samples were treated with TCA (Trichloroacetic Acid) in order

to precipitate out the proteins from the solutions away from the ions. The protein

precipitate was then extracted and then carried to lowry’s method. Though there is a need

for dilution of the samples, as the absorbance was high than 1-1.2 due to high protein

content. Lowry’s method works fine for lower concentrations of proteins.

SDS PAGE gels: The oldest cultures with tellurite salt showed a unique phenomenon, in which the

colour of the media turned from black to light green, after incubation for longer time. The

change of media colour into black is due to synthesis of metal nanoparticles, but the change

into light green colour indicate that the metal nanoparticles are converted into some other

form from the solution. Probably into volatile gas compounds of tellurium (as it was

previously reported that few marine bacteria show this phenomenon)

It was also observed that, more the headspace in the flask, faster will be the

reduction of metal and later into volatile compounds.

Though few bands were observed differentially in the oldest cultures with tellurite

salt, it couldn’t be confirmed as the protein loading was not even in all the wells.

Conclusion: Protein Profiles: To avoid uneven loading of protein samples into the wells,

Lowry’s methods need to be performed prior to SDS PAGE. Depending on the

resulting concentrations in each sample, they might have to be either concentrated or

diluted and load equal amount of protein in all wells.

The Standard Graph For Lowry’s Method:

The standard graph for the lowry’s method was prepared. The protein estimation of

the samples can be done before loading into SDS PAGE wells.

More the headspace, faster was the reduction of Tellurite into tellurium metal

Nanoparticles.

The TeNPs are getting converted into some volatile compounds as the is incubated for

longer time and it is faster if the headspace is more.The same need to be tested against

Selenium salt to assess the bacteria’s reaction towards exposure of metal salt for

longer time.

y = 0.0003x - 0.0163

0

0.01

0.02

0.03

0.04

0.05

0.06

0 50 100 150 200 250

Ab

sorb

ance

Concentration μg/ml

Concentration.vs.absorbance

Concentration.vs.absorbance

Linear(Concentration.vs.absorbance)

Bibliography: Volatilization and Precipitation of Tellurium by Aerobic, Tellurite-Resistant

Marine Microbes Patrick R. L. Ollivier, Andrew S. Bahrou, Sarah Marcus, Talisha

Cox, Thomas M. Church, and Thomas E. Hanson. 2008

The Lowry Method for Protein Quantitation Jakob H. Waterborg

Thermo Scientific Pierce Electrophoresis Technical Handbook, Version 2