. .
.
Experiment-2: Gel-based proteomics to analyze bacterial proteome
.
.


BACTERIAL PROTEOMICS

 

Escherichia coli is one of the most widely used model organisms in the world, which has paved the way for a new era of biotechnology and related sciences. E. coli is a prokaryotic, unicellular microorganism that can easily be grown, harvested and manipulated in order to understand various biological processes. Many landmark discoveries in biology have been made using E. coli as the model and the same theory has been successfully extrapolated to higher organisms.

The proteome refers to the entire protein complement expressed by the genome of an organism at a given point of time under a defined set of conditions. Since proteins are the ultimate effector molecules in all organisms, study of E. coli proteins could provide important insights into various biological functions.  There are several methods to study the numerous proteins being expressed by E. coli, many of which are laborious and time consuming. The gel-based proteomics approach, which has increasingly gained popularity to study large number of proteins simultaneously, will be discussed in detail in the following sections.

Culturing of E. coli.

  All glassware and media to be used for growing the bacteria are first autoclaved at 15 lb pressure for 15 minutes at 1210C.

   2% Luria broth is prepared in a clean autoclaved container.

   The broth is then inoculated with around 100 ul of fresh culture.

   The culture is maintained at 370C, with constant agitation in an incubator for about 6-8 hours.

   Proper growth of the microbe can be ensured by measuring the optical density of the culture solution at regular time intervals. The O.D value should ideally be between 0.8 and 1.0.

 Once the culture reaches the desired O.D, the organism’s growth is stopped by centrifuging the culture in tubes at 5000 g for 15 minutes at 40C.

 

 

 

Fig 17 : a) Picking organism colonies from the mother culture b) Inoculation of the culture broth c) Monitoring growth of E. coli by turbidity measurements d) Precipitation of bacterial pellet using centrifugation.

 

Extraction of proteins from E. coli

Unlike serum, the entire E. coli organism is considered for studying its proteome. Hence methods are required to lyse the cells and release its cytoplasmic contents, including the various proteins to be studied. Cell lysis can be carried out at the lab scale with the help of chemical and physical methods, of which sonication is one of the most widely used and accepted techniques. The procedure for sonication and subsequent protein extraction is explained below.

 

 

 The cells must first be harvested out of the broth, which is done by centrifuging the culture at 13,000g for 15 minutes. This causes the cells to sediment out in the form of a pellet.

  The pellet containing the E. coli cells is washed 3 times with 1 mL of  phosphate buffer, pH 7.4 (20mM Na-phosphate, 0.15M NaCl).

  The washed pellet is then re-suspended in a re-suspension solution, pH 7.4 (20mM Na-phosphate, 0.15M NaCl, and 5mM MgCl2). To this, 10 uL/mL of protease inhibitor is added, which ensures that there is not degradation of the proteins when they are released along with the protease enzymes.

  This mixture is sonicated, which breaks open the cells in the pellet, thereby releasing all the cytoplasmic contents. Sonication is performed 3 times for 30 cycles at 40% amplitude. 

Sonicated bacterial protein suspension is subjected to protein precipitation procedure using TRIzol protocol.

 

Trizol (contains phenol and guanidine isothiocyanate) method helps for simultaneous extraction of RNA, DNA and proteins. After addition of Trizol all 3 components separates into 3 distinct layers. Proteins is extracted at the end by the addition of acetone (for precipitation) to the lower most portion of layers.

    Add 1 ml trizol reagent to the bacterial suspension.

    Immediately add 200ul chloroform to the same mixture, shake vigorously for 15 sec and incubate for 15 min at RT.

    Centrifuge at 12,000g for 15 min.

    Carefully remove upper layer containing RNA using a micropipette.

    To the bottom layer, add 300 ul ethanol, centrifuge at 5,000g for 5 min to remove DNA.

    Separate the supernatant containing protein collect into a fresh tube. Retain the pellet of DNA.

    To the resultant supernatant, add 4 volumes of chilled acetone (acetone kept in – 200C at least for 4 hours) and incubate for 6 hrs at – 200C.

    After incubation centrifuge at 12,000g for 5 min.

    Discard the supernatant, retain the pellet of protein.

    Wash the protein pellet with 95% (95% ethanol+5% water) ethanol or acetone (4 times).

    After wash give a brief spin to settle proteins. Every time through the supernatant.

    Dry the pellet in the room temperature.

    Reconstitute the dried pellet in lysis buffer.

 

 

 

 

Fig 18: Image of a typical gel showing bacterial proteins separated on a 4-7 pH range over X axis and Molecular weight on Y axis.

 

 

Experiment-2: Gel-based Proteomics to Analyze Bacterial Proteome

 

 

 

OBJECTIVE:
To compare the protein expression profiles of E. coli cultures grown under normal and drug treated conditions using 2D gel electrophoresis.
 
THEORY
Escherichia coli is one of the most widely used model organisms for studying prokaryotic systems, which has paved the way for a new era of biotechnology and related sciences. E. coli is a prokaryotic, unicellular microorganism that can be easily grown, harvested and manipulated in order to understand various biological processes. Many landmark discoveries in biology have been made using E. coli as the model and the same theory has been successfully extrapolated to higher organisms. The proteome refers to the entire protein complement expressed by the genome of an organism at a given point of time under a defined set of conditions. Proteomics describes the comprehensive study and characterization of complete set of proteins of an organism at a given time. Since proteins are the ultimate effector molecules in all organisms, study of E. coli proteins could provide important insights into various biological functions. There are several methods to study the numerous proteins expressed in E. coli, many of which are laborious and time consuming. The gel-based proteomics approach, which has increasingly gained popularity to study large number of proteins simultaneously, will be discussed in detail in the following sections. Two-dimensional gel electrophoresis (2DE) is one of the most widely used electrophoretic techniques for separation of complex mixtures of proteins due to its ease of use. Protein separation is carried out based on two different properties of proteins namely, isoelectric point and molecular weight. For detail theory of 2DE users are advised to see the Experiment 1.
 
Culturing of E. coli
  1. All glassware and media to be used for growing the bacteria culture are first autoclaved at 15 lb pressure for 15 min at 121°C.
  2. 2% Luria broth is prepared in a clean autoclaved container.
  3. The broth is then inoculated with around 100 μL of fresh E. coli culture.
  4. The culture is maintained at 37°C, with constant agitation in an incubator for about 6-8 h.
  5. Proper growth of the microbe can be ensured by measuring the optical density of the culture solution at regular time intervals. The OD value should ideally be between 0.8 and 1.0.
  6. Once the culture reaches the desired OD, the organism’s growth is stopped by centrifuging the culture at 5000 g for 15 min at 4°C.

 

                                                   

                                                            

 

 

Figure 1.  Different steps involved in culturing of the E. coli; (a) pick the organism colonies from the mother culture (b) inoculate the culture into the fresh broth (c) monitoring growth of E. coli by turbidity measurements and (d) stopping the growth and precipitation of bacterial pellet by centrifugation.
 
 
Extraction of proteins from E. coli
For studying E. coli proteome methods are required to lyse the cells to release its cytoplasmic contents to include entire proteome. Cell lysis can be carried out using different chemical and physical methods, among which sonication is one of the most widely accepted, effective and easy approach. Procedure for sonication and subsequent protein extraction is explained below.
 
  1. The cells must be harvested out of the broth, which is done by centrifuging the culture at 13,000 g for 15 min. This causes the cells to sediment out in the form of a pellet.
  2. The pellet containing the E. coli cells is washed 3 times with 1 mL of  phosphate buffer of pH 7.4 (20 mM Na-phosphate, 0.15 M NaCl).
  3. The washed pellet is then re-suspended in a re-suspension solution of pH 7.4 (20 mM Na-phosphate, 0.15 M NaCl, and 5 mM MgCl2). To this 10 μL/mL of protease inhibitor is added to prevent protein degradation by proteolytic enzymes.
  4. This mixture is subjected to mild sonication, which breaks open the cells, thereby releasing all the cytoplasmic contents. Sonication is performed 3 times for 30 cycles at 40% amplitude. 
  5. Sonicated bacterial protein suspension is subjected to protein precipitation procedure using Trizol reagent.
 
Trizol reagent contains phenol and guanidine isothiocyanate which helps in simultaneous extraction of RNA, DNA and proteins. This is particularly a liquid-liquid phase extraction, where DNA, RNA and protein get separated into 3 distinct layers depending on their solubilization properties. The layer containing the protein content is treated with acetone for precipitation and pellet formed can be stored for further use. 
 
1.       Add 1 mL trizol reagent to the bacterial suspension.
2.       Add 200μL chloroform to the same mixture immediately, shake vigorously for 15 sec and incubate for 15 min at RT.
3.       Centrifuge at 12,000 g for 15 min to form the layers for separation.
4.       Carefully remove upper layer containing RNA using a micropipette without disturbing the other layers.
5.       To the bottom layer, add 300 μL ethanol, centrifuge at 5,000 g for 5 min to remove DNA.
6.       Separate the supernatant containing protein and collect into a new tube. Retain the pellet of DNA.
7.       To the resultant supernatant, add 4 volumes of chilled acetone (acetone kept in – 20°C at least for 4 h) and incubate for 6 h at – 20°C.
8.       After incubation centrifuge at 12,000 g for 5 min.
9.       Discard the supernatant, retain the pellet of protein.
10.    Wash the protein pellet with 95% (95% ethanol+5% water) ethanol or acetone (4 times).
11.    After washing give a brief spin to settle down the proteins. Each time discard the supernatant without disturbing the pellet.
12.    Dry the pellet at room temperature.
13.    Reconstitute the dried pellet in lysis buffer for further use.

 

 
 
                                                   

 

Figure 2. Image of a typical 2D gel showing bacterial proteome separated on a 4-7 pH range IPG strip.

 

 

Cite this Simulator:

.....
..... .....
Copyright @ 2017 Under the NME ICT initiative of MHRD (Licensing Terms)
 Powered by AmritaVirtual Lab Collaborative Platform [ Ver 00.11. ]