LABORATORY ORIENTED PROJECT

A report on:

Biosynthesis of Tellurium
Nanoparticles and Protein Profile
Studies on Marine Bacteria
Submitted to

Dr. Meenal Kowshik

Associate 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
Lowrys method: ........................................................................................................................... 13
Results & Discussions: ........................................................................................................................... 15
Lowrys Method: ........................................................................................................................... 15
SDS PAGE gels: .............................................................................................................................. 15
Conclusion: ............................................................................................................................................ 16

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

The Standard Graph For Lowrys 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 nanoclusters.
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 37 0C for
40 hr, and their growth is observed.

Te
(mM)
0.05
0.1
0.25
0.5
0.1
Cntrl

Inoculum
(L)
200
200
200
200
200
0

Tellurite
(L)
10
20
50
100
200
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 30m) 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:

Lowrys method:
The lowrys 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 g/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:

Lowrys 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 lowrys 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. Lowrys 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 couldnt 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,
Lowrys 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 Lowrys Method:

Concentration.vs.absorbance
0.06

Absorbance

0.05

y = 0.0003x - 0.0163

0.04
0.03

Concentration.vs.absorbance

0.02

Linear
(Concentration.vs.absorbance)

0.01
0
0

50

100

150

200

250

Concentration g/ml

The standard graph for the lowrys 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 bacterias reaction towards exposure of metal salt for
longer time.

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