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Experiment-3: Gel-based proteomics to analyze plant proteome
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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-3: Gel-based Proteomics to Analyze Plant Proteome

 

   

OBJECTIVE
To compare the protein expression profiles of plant leaves grown under drought (stress) and normal conditions using 2D gel electrophoresis.
 
THEORY 
 
Plants have appreciable amount of complexity with respect to protein networks. Studying the proteome of plants could help in providing a comparative analysis with closely related proteomes of other organisms. Some plants such as Arabidopsis thaliana, which is commonly used as a model organism, are often studied at the proteome level to gain deeper insights into the various biological processes. Some plants have therapeutic values and are often studied to get more information of the protein nature, when exposed to several stress responses. In this section, we have described the procedure for analysis of plant leaf proteome using 2DE. Experimental plan involves the collection of plant leaf, protein extraction followed by separation of proteins on 1st and 2nd dimension. For detail theory of 2DE users are advised to see the Experiment 1.
 
 Preparation of sample:
  1. Around 300 mg of fresh leaf is collected.
  2. First of all the leaves must be homogenized properly using a clean mortar and pestle.
  3. Liquid nitrogen is added to the homogenized leaves to freeze and solidify rapidly. This solidified leaf parts are then mashed thoroughly until a powder is obtained.
  4. To the powder, 500 µL of lysis buffer (acetone, 10% TCA, 0.07% DTT) is added and the contents are mashed vigorously to prepare a homogenous mixture. More lysis buffer can be added, if required, to make the process efficient. Once it is finely homogenized, the volume is made up to 1.5 mL and this mixture is kept at -20°C for 1 h.
  5. It is then centrifuged at 14,000 g for 30 min at 4°C. This enables the formation of a pellet of proteins and the supernatant can be discarded.
  6. Chilled acetone with 0.07% DTT is then added to the pellet and vortexed briefly.
  7. The mixture is again centrifuged at 14,000 g for 30 min at 4°C, after which the supernatant is discarded. This step is repeated 3 more times.

 

                                                   

                     

Figure 1: Protein extraction from the plant leaves. (a) Fresh leaves, clean the leaves properly (b) treat the leaves with liquid nitrogen and start grounding (c) fine homogenous paste prepared using buffer (d) debris precipitated using centrifugationn                           

 

  1. After washing step, the pellet is left to dry at room temperature for about 40 min. Ensure that the pellet is totally dry.
  2. The dry pellet is then dissolved in 400 µL of rehydration buffer (composition same as mentioned in section 1: C) and vortexed briefly. This is then stored overnight at 4°C for protein extraction.
  3. Next day, the mixture is centrifuged at 14,000 g for 15 min at 4°C.
  4. The supernatant containing the proteins are separated carefully and stored in a fresh microcentrifuge tube at -20°C until further use.
 

 

 
 
                                                  

 

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

 

 

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