LAB 6: BACTERIAL DNA EXTRACTION

Introduction

DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms. The information in DNA is stored as a code made up of four chemical bases which are adenine (A), guanine (G), cytosine (C), and thymine (T). The order of these bases determines the information available for building and maintaining an organism. The DNA of most bacteria is contained in a single circular molecule, called the bacterial chromosome. In addition to the chromosome, bacteria often contain plasmids which is a small circular DNA molecules. Plasmids are usually used to purify a specific sequence since they can be easily be purified away from the rest of the genome. So, plasmids need to be isolated for their use as vectors and also for molecular cloning. 

There are two large groups of bacteria based on their different cell wall constituents, which are gram-positive and gram-negative bacteria. Gram-positive bacteria are bacteria that give a positive result in the Gram stain test. They appear to be purple colored when seen through a microscope due to the thick peptidoglycan layer in the bacterial cell wall which retains the stain after it is washed away from the rest of the sample, in the decolorization stage of the test. Examples are Lactobacillus fermentum, Bacillus subtilis, Streptococcus agalactiae. Gram-negative bacteria cannot retain the violet stain after the decolorization step; alcohol used in this stage degrades the outer membrane of gram-negative cells making the cell wall more porous and incapable of retaining the crystal violet stain. Their peptidoglycan layer is much thinner and sandwiched between an inner cell membrane and a bacterial outer membrane, causing them to take up the counter strain and appear as red or pink. Examples are Escherichia coli and Salmonella typhimurium .

DNA extraction is a routine procedure used to isolate DNA from the nucleus of cells. Extraction is an easy and quick way to purify DNA from a mixture of proteins, lipids and nucleic acids. The reason for this procedure is to separate the plasmid DNA from its associated proteins so that further manipulations can be done to it. DNA extraction involves several processes such as lysozyme treatment, centrifugation, protein denaturation, homogenization etc. The lysozyme treatment is important to break down bacterial cell walls to improve nucleic acid extraction efficiency.

The GF-1 Bacterial DNA Extraction Kit is designed for rapid and efficient purification up to 20μg of a high molecular weight genomic DNA from 1-3 ml either Gram-negative or Gram–positive bacteria. This bacterial purification kit applies the principle of a minicolumn spin technology and the use of optimized buffers to ensure that only DNA is isolated while cellular proteins, metabolites, salts and other low molecular weight impurities are removed during the subsequent washing steps.

Figure 1: GF-1 Bacterial DNA Extraction Kit

DNA concentration and purity can be assessed using absorbance (optical density) of DNA solution. The intensity of absorbance of the DNA solution is measured at wavelengths 260 nm ad 280 nm. DNA absorb ultraviolet (UV) light due to the heterocyclic rings of the nucleotides; the sugar-phosphate backbone does not contribute to absorption. To ensure the concentration reading is accurate, the absorbance reading should be within the linear range of the spectrophotometer. The Lambert-Beer law relates the absorption of light to the properties of the material through which the light is travelling. Several factors that can influence the accuracy of A260/A280 ratio includes type of protein present, absorbance of phenol and other contaminants at 280 nm and pH of DNA solution.

Objective

- To understand and practice proper technique of isolation process of plasmid DNA from bacteria.

- To determine the DNA concentration using Beer-Lambert equation

Materials and Apparatus

Columns 

Collection tubes 

Receiver

Pipette

Incubator (Digital Dry Bath)

Centrifuge

Spectrophotometer

Microcentrifuge tube

Gram-negative bacteria strain, Escherichia coli

Gram-positive bacteria strain, Lactobacillus fermentum
Resuspension Buffer 1
Resuspension Buffer 2

Lysozyme
Proteinase K solution 

Ethanol
Wash Buffer 
Elution Buffer 

Procedure:

Reminder:

* All steps are to be carried out at room temperature unless stated otherwise.

* Wash buffer (concentrate) has to be diluted with absolute ethanol before use.

* Pre-heat Elution Buffer to 65°C (optional).

1. Centrifugation. 1ml of bacteria culture grown overnight or culture grown to log phase is turned into pellet by centrifugation at 6000 x g for 2 minutes at room temperature. The supernatant is decanted completely.

2. Resuspension of pellet. 100µl Buffer R1 is added to the pellet and the cells are resuspended completely by pipetting up and down.

3. Lysozyme treatment. For Gram-negative bacteria strain (Escherichia coli), 10 µl lysozyme (50mg/ml) is added into the cell suspension. For Gram-positive bacteria strain (Lactobacillus fermentum), 20 µl lysozyme (50mg/ml) is added into the cell suspension. They are mixed thoroughly and incubated at 37°C for 20 minutes.

4. Centrifugation. Digested cells are centrifuged at 10000 x g for 3 minutes and pellet is formed. The supernatant is decanted completely.

5. Protein denaturation. Pellet is resuspended in 180 µl of Buffer R2 and 20 µl of Proteinase K is added. It is mixed thoroughly. Then, it is incubated at 65 °C for 20 minutes with occasional mixing every 5 minutes.

6. Homogenization. 400 µl without RNase A treatment of Buffer BG is added and is mixed thoroughly by inverting tube several times until a homogenous solution is obtained. It is incubated for 10 minutes at 65°C.

7. Addition of ethanol. 200 µl of absolute ethanol is added, then is mixed immediately and thoroughly.

8. Loading to column. The sample is transferred into a column assembled in a clean collection tube. It is centrifuged at 10000 x g for 1 minute. Flow through is discarded.

9. Column washing. The column is washed with 650 µl of Wash Buffer and is centrifuged at 10000 x g for 1 minute. Flow through is discarded.

10. Column drying. The column is centrifuged at 10000 x g for 1 minute to remove residual ethanol.

11. DNA elution. The column is placed into a clean microcentrifuge tube. 50 µl of pre-heated Elution Buffer, TE buffer is added directly onto column membrane and is allowed to stand for 2 minutes. It is centrifuged at 10000 x g for 1 minute to elute DNA.

12. The DNA content is measured using spectrophotometer with absorbance at 260nm and 280nm.

Figure 2: Gram-positive bacteria (Lactobacillus fermentum) and Gram-negative bacteria (Escherichia coli).

Figure 3: Centrifugation.

Figure 4: Resuspension.

Figure 5: Incubation using digital dry bath.

Figure 6: DNA content is measured using spectrophotometer with absorbance at 260nm and 280nm.

Result

Table 1: A260/A280 ratio of the samples

Bacteria	Optical Density		Ratio (260/280)
	260	280
Escherichia Coli (Gram-negative)	0.069	0.035	1.97
Lactobacillus fermentum (Gram-positive)	0.264	0.157	1.681

The DNA concentration of the bacteria is calculated by using Beer-Lambert equation shown below:

 A=ƸCl

A= A260 (absorbance) Ƹ=0.020 (molar extinction coefficient) C= concentration of DNA l= 0.051 (light pathlength)

                                                  

For the DNA concentration of Escherichia Coli (Gram-negative),

Let C= DNA concentration of Escherichia Coli

By using Beer-Lambert equation,

A=ƸCl 0.069= 0.020 (C) (0.051) C=0.069/(0.020x0.051)  C= 67.64 µg/ml

For the DNA concentration of Lactobacillus fermentum (Gram-positive),

Let C= DNA concentration of Lactobacillus fermentum

By using Beer-Lambert equation,

A=ƸCl 0.264= 0.020 (C) (0.051) C=0.204/(0.020x0.051)  C= 258.82 µg/ml

Discussion

     Before discussing the result, a brief explanation on the Gram-positive bacteria, Gram-negative bacteria, lysozyme, precaution and others are discussed. Gram staining is a technique developed by Christian Gram in 1884 which is used to strain bacteria. The bacteria which retain the colour of the stain are called Gram-positive bacteria whereas the bacteria which lose the colour of the stain are called Gram-negative bacteria. Gram-positive cell is bacterial cell having wall composed of thick layer of peptidoglycan containing teichoic acids and retaining the crystal violet dye used in the Gram staining procedure, appearing purple when seen through a microscope. Gram-negative cell is bacterial cell having a wall composed of a thin layer of peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane, besides that it do not retain the crystal violet stain used in the Gram staining method.

    Lysozyme is an enzyme that destroy the bacterial cell walls by catalytic hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan.

        

     In this experiment, some precaution steps must be taken. For instance, when re-suspend the cell completely by pipetting up and down, place the pipette on the same position in order to make sure complete cell re-suspension. Besides, when centrifuge, place the column which has a triangle marks on the edge to ensure that the process achieves optimization.

       

   OD260 unit, is a spectrophotometric measurement of an oligonucleotide. It is a normalized unit of measurement that is defined as the amount of oligonucleotide required to give an absorbance reading of 1.0 at 260 nm in 1.0 mL of solution using a 1 cm light path. Each of the bases in a nucleic acid strand has an absorbance at or near 260 nanometers, due to their conjugated double bond systems. Because the exact base sequence and composition is known, the OD260 unit is a precise method to quantify an oligonucleotide. Utilizing absorbance measurements is the recommended method for quantitating or aliquotting an oligonucleotide. Absorption at 280 nm indicates the presence of proteins due to the strong absorption by the aromatic amino acids.

           

     Ratio between the readings at 260 nm and 280 nm (A260/A280) provides an estimate of the purity of the DNA. A ratio of approximately in the range of 1.7-1.9 is generally accepted as “pure” for DNA. If the ratio is lower than this range, the DNA cannot be considering as “pure”. There are few factors that cause the low DNA ratio reading, one of that is caused by protein or phenol contamination that absorb strongly at or near 280 nm. Moreover, other factors include incomplete cell suspension, low elution efficiency, column clogged and incomplete protein denaturation.

            

     From the result obtained, the ratio of A260 to A280 for Escherichia Coli is 1.97. This indicates the ratio of DNA in the extract is not high enough to be considered as “pure”. This value is slightly higher than the range of 1.7 to 1.9. This deviation may due to the contamination by the presence of RNA. Mild alkaline condition may also contribute to the increase in the ratio of A260/A280.

            

     The ratio of A260 to A280 for Lactobacillus fermentum is 1.68. The value calculated is slightly lower but very close to the range of 1.7 to 1.9 which means that the materials that are extracted are mainly DNA with little contamination by other materials like proteins.

           

     The concentration of DNA in E.coli obtained from the result is 67.64 µg/ml while the concentration of DNA in L.fermentum is 258.82 µg/ml. It is clearly shown that more DNA are extracted from L.fermentum. This may be caused by the technical errors done in the procedure of extracting the DNA from E.coli, resulting in lower level of DNA extracted. We can also compare the readings of DNA concentration of each bacteria with the ratios of OD260/OD280 respectively. For E.coli, the ratio obtained is 0.07 higher than the range where “pure” DNA is extracted. On the other hand, for L.fermentum, the ratio is only 0.02 lower than the range. Thus, the concentration of DNA extracted is higher in L.fermentum than E.coli.

Conclusion

DNA extraction requires a proper method and technique in order to obtain high DNA concentration. The bacterial extract containing DNA is placed in a spectrophotometer to measure the absorbance using light wavelengths at 260 nm and 280 nm. The ratio of A260/A280 gives the estimate of the DNA purity in the extract. The best ratio that contains highest DNA purity is between 1.7 to 1.9. Then, Beer-Lambert equation is used to calculate the concentration of DNA.

Reference

https://www.promega.com/-/media/files/resources/application-notes/pathlength/calculating-nucleic-acid-or-protein-concentration-using-the-glomax-multi-microplate-instrument.pdf?la=en

http://www.ogt.com/resources/literature/483_understanding_and_measuring_variations_in_dna_sample_quality

LAB 5: DETERMINATION OF ANTIMICROBIAL EFFECTS OF MICROBIAL EXTRACTS

Introduction

           An antimicrobial is an agent that kills or inhibits the growth of microorganisms. The microbial agent may be a chemical compounds and physical agents. These agents interfere with the growth and reproduction of causative organisms like bacteria, fungi, parasites, virus etc. Antimicrobial substances can be produced by certain group or species of bacteria with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. For instance, the microbial compounds include organic acids, hydrogen peroxide, diacetyl and bacteriocins. The use of these substances with antimicrobial properties is known to have been common practice for at least 2000 years. Antimicrobial agents are widely used in food system and resistance management which plays a role in controlling both pathogenic and spoilage microbe to grow. Nowadays, consumers demand “natural” and “minimally processed” food, the interest in naturally produced antimicrobial agents such as bacteriocins is on the rise of demand. The discovery of bacteriocins gave a new way for food development in better hygienic quality.

Bacteriocins are a kind of ribosomal synthesized antimicrobial peptides produced by bacteria, which can kill or inhibit bacterial strains closely-related or non-related to produced bacteria, but will not harm the bacteria themselves by specific immunity proteins. Bacteriocins become one of the weapons against microorganisms due to the specific characteristics of large diversity of structure and function, natural resource, and being stable to heat. Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and mechanism of killing. Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and mechanism of killing. Moreover, bacteriocins can be found in numerous Gram-positive and Gram-negative bacteria, those produced by lactic acid bacteria (LAB) have received special attention in recent years due to their potential application in the food industry as natural biopreservatives.

LAB also known as Lactobacillalesare either rod-shaped (bacillus), or spherical (coccus).Different classes of LAB bacteriocins have been identified on the basis of biochemical and genetic characterization. These bacteriocins have been reported to inhibit the growth of Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum. In this experiment, Salmonella bacteria and Escherichia coli are used.

Objective

To determine the antimicrobial effects of extracelluar extracts of selected LAB strains

Materials and reagents

MRS broth

Sterile filter paper disk (50mm x 50mm)

Forceps

Sterile universal bottles

Cultures of LAB and spoilage/ pathogenic organisms (Escherichia coli and Salmonella )

Bench-top refrigerated centrifuge

Incubator 30°C and 37°C

UV/Vis spectrophotometer

Distilled deionized water

Trypticase soy agar

Brain heart infusion agar

Yeast extract

Bunsen burner

Pipette and tips

Sterile petri dish

96 well plate

Procedure

Part 1: Determination of bacteriocin activity via agar diffusion test

1. All the petri dishes are labelled according to the spoilage organisms and strains of LAB used.

2. Each plate are used for one strain of spoilage organism and one strain of LAB by dividing the plate into 2 sides, each side for one replicate.

3. 2 strains of LAB and 2 strains of spoilage/ pathogenic organisms are given to each group.

4. 10 ml of trypticase soy-yeast extract agar (TSAYE) are loaded into the labelled petri dishes using pipette and “ figure of 8 ” is performed to ensure that the entire surface of the plate is covered by the agar. The petri dishes were left aside for the agar to be solidified.

5. 2 ml of the broth containing the spoilage organism are innoculated into 10 ml of brain heart infusion (BHI) agar and the misture are vortexed.

6. The mixture is then quickly loaded on top of the TSAYE agar layer, it is ensured to cover the entire surface and the petri dishes are left aside for the mixture to solidify.

7. The broth containing LAB cultures are centrifuged and the supernatant obtained are used as the extracellular extracts by draining off the excess extract.

8. A sterile filter paper disk is picked up aseptically using sterile forceps and the disk is dipped into the extracellular extract.

Figure 1: A sterile filter paper disk is picked up aseptically using sterile forceps 

Figure 2: The disk is dipped into the extracellular extract. 

9. The paper disk is placed on top of the solidified BHI agar.

Figure 3: The disk are placed on top of the agar. 

10. The plates are incubated for 24-48 hours at 37°C.

Figure 4: The plates are placed into incubator.

11. The inhibition zones (in cm) is measured after incubation and the readings are recorded.

Part 2: Determination of bacteriocin activity via optical density

1. The broth containing LAB cultures are centrifuged. The supernatant are used as the extracellular extracts.

2. Each group are given 2 strains of LAB and 2 strains of spoilage/pathogenic organisms.

3. 10 µl of double-strength MRS are added with 10 µl of cultures containing spoilage/ pathogenic organism and the mixture is vortexed.

4. A serial dilution of LAB extracellular extract with MRS was prepared with final volume of 1000 μl in each column. The volume of the LAB extracellular extract and MRS needed for each column is shown as below:

Table 1: Serial dilution

Mixture	Dilution
	0x	2x	10x	50x	100x	Control
LAB Extracellular Extract (μl)	1000	500	200	200	500	0
MRS Broth (μl)	0	500	800	800	500	1000
Total (μl	1000	1000	1000	1000	1000	1000

Figure 5: LAB extracellular extract is loaded according to the volume required.

Figure 6: MRS broth is added to the LAB extracellulat extract.

Figure 7: Serial dilution is done.

5. 50 μl of each extracellular extracts dilution are added into mixture as prepared in step 3.

Figure 8: The extracellular extracts dilution are loaded into 96 well plate.

6. The mixtures are incubated for 12-15 hours at 37⁰C.

7.  A control is  prepared using 5 ml of double-strength MRS, 1 ml of cultures containing spoilage/pathogenic bacteria and 10 ml of MRS. The mixtures are incubated for 12-15 hours at 37⁰C.

8.  A negative-control is prepared for “auto-zero” via spectrophotometer. 5 ml of double-strength MRS are added to the control.

Figure 9: Spectrophotometer

9. Upon incubation, the optical density of the spoilage/pathogenic bacteria are measured at 600 nm. The same method is performed for the control as well.

10.  One arbitrary unit (AU) is defined as the dilution factor of the extracellular extract that inhibited 50% of the spoilage/pathogenic bacteria growth and expressed as AU/ml.

11. 50% of the spoilage/pathogenic bacteria growth are determined from the OD₆₀₀ of the control.

Results

Part I: Determination of bacteriocin activity via agar diffusion test

Figure 10: Petri dish containing LAB 1 and E.coli

Figure 11: Petri dish containing LAB 1 and Salmonella  

Figure 12: Petri dish containing LAB 2 and E.coli

Figure 13: Petri dish containing LAB 2 and Salmonella

Table 1: Inhibition zone of strains of LAB on strains of spoilage/pathogenic bacteria

Strains of LAB	Strains of spoilage/ pathogenic bacteria	Inhibition zone (cm)
LAB 1	E.coli	1.0
	Salmonella	0.6
LAB 2	E.coli	0.8
	Salmonella	0.6

Part II: Determination of bacteriocin activity via optical density

Serial dilution of extracellular extract

LAB 1

Figure 14: 96 well plate containing E.coli and Salmonella 

with different dilutions of LAB 1

Table 2: OD₆₀₀ of E.coli according to different dilutions of LAB 1

Dilutions	OD600 of spoilage/ pathogenic bacteria
	Strain 1: E.coli
	Reading 1	Reading 2	Reading 3	Average
0x	0.263	0.436	0.433	0.3773
2x	0.712	1.399	1.389	1.1667
10x	0.835	1.436	1.491	1.254
50x	0.792	0.963	1.415	1.0567
100x	0.582	0.819	1.102	0.834
Equation	y = 0.0803x + 0.6967
OD600 of control	0.582	0.819	1.102	0.834
50% of OD600	0.291	0.410	0.551	0.417
AU/ml	X= (0.417-0.697)/0.0803    = -3.487

Graph 1: OD₆₀₀ of E.coli against different dilutions of LAB 1

Table 3: OD₆₀₀ of Salmonella according to different dilutions of LAB 1

Dilutions	OD600 of spoilage/ pathogenic bacteria
	Strain 2: Salmonella
	Reading 1	Reading 2	Reading 3	Average
0x	0.329	0.323	0.159	0.2703
2x	1.153	1.324	0.790	1.089
10x	1.468	1.476	0.961	1.3017
50x	1.428	1.340	0.920	1.2293
100x	0.956	0.997	0.681	0.878
Equation	y = 0.1356x + 0.547
OD600 of control	0.956	0.997	0.681	0.878
50% of OD600	0.478	0.499	0.341	0.439
AU/ml	X= (0.439-0.547)/0.1356    =-0.796

Graph 2: OD₆₀₀ of Salmonella against different dilutions of LAB 1

Discussion

Part I: Determination of bacteriocin activity via agar diffusion test

      Bacteriocins are protein which is secreted by bacteria to inhibit the growth of other closely- related bacterial strain. Bacteriocin causes the destruction of the membrane potential, forming the pores on the pathogenic bacteria. Bacteriocin also inhibits the protein synthesis of the pathogenic bacteria. Besides that, it inhibits the nucleolytic activity of the pathogenic bacteria strains which breaks down the DNA chromosomes as well as RNA. In this experiment, bacteriocin is produced by lactic acid bacteria (LAB).

     Lactic acid bacteria are rod-shaped bacilli or cocci characterized by an increase tolerance to a lower pH range. The production of organic acids such as lactic acid and acetic acid by lactic acid bacteria causes acidification, hence it can also be used to inhibit spoilage bacteria.

Bacteriocin are usually effective against Gram-positive bacteria. Although Gram-positive bacteria has thick cell wall made of protein and polysaccharides, but is easily digested by acid produced by lactic acid bacteria (LAB). LAB may be not efficient enough to inhibit Gram-negative bacterium because the cell wall is made from lipid layer which prevent acid from being digested by acid produced by LAB. In this experiment, the spoilage bacteria that we used are Escherichia coli and Salmonella, in which both are Gram-negative bacteria.

     In agar diffusion test, a filter-paper disk saturated with bacteriocin of LAB is placed on the surface of the agar. The compound diffuses from the filter paper into the agar. The concentration of the compound near the disk would be the highest, and will decreases as distance from the disk increases. The effectiveness of LAB antimicrobials towards the growth of spoilage microorganisms can be determined by measuring the inhibition zones around the LAB-staining paper disks. Inhibition zone is the clear region around the paper disc, which is an indication of the absence or the effective inhibition of microbial growth by the antimicrobial agent. The larger the inhibition zones, the higher the degree of sensitivity of spoilage microorganisms to LAB antimicrobials.

     As a result, the average measurement of inhibition zone for Escherichia coli is 0.9cm while for measurement of inhibition zone for Salmonella is 0.6cm. The inhibition effect of LAB is greater towards Escherichia coli compared to Salmonella.

Part 2: Determination of bacteriocin activity via optical density

     Optical density is a measurement of the concentration of bacteria in a suspension, which can be measured using a spectrophotometer. Spectrophotometer is an instrument which can be used to measure the amount of light scattered at a specific wavelength when passes through a medium. When visible light passes through a cell suspension, the light is scattered. Greater degree of scatter indicates that more bacteria are present. A spectrophotometer can be set at a wavelength of 420 – 660 nm. In this experiment, the OD600 is measured. OD600 is an abbreviation indicating the optical density of a sample is measured at a wavelength of 600 nm, which is much preferable because at this wavelength, the cells will not be killed due to the exposure of too much light intensity.

One arbitrary (AU) is known as the dilution factor of the extracellular extract that inhibited 50% of the spoilage or pathogenic bacteria growth and expressed as AU/ml.

Control: Abs600 = Z. Thus, 50% of Z = Z/2 

y = mx + c; Thus, x = (y-c)/m

When y = Z/2, Thus x = (Z/2 -c)/m

Both graph plotted from the data we obtained has positive gradient from 0x to 10x dilution, which means LAB 1 shows positive inhibition on Escherichia coli and on Salmonella. The lower the concentration of extracellular extract, the lower concentration of bacteriocin, the higher the growth rate of bacteria. However, the graphs later show negative gradient from 10x to 100x dilution. There is a decreasing trend on the growth of bacteria from 10x to 100x dilution. The result obtained might not be accurate due to the improper preparation of the serial dilution solution. This causes the result obtained is not like what it is supposed to be. This means that both graph should always have positive gradient from 0x to 100x dilution.

Conclusion

Lactic acid bacteria (LAB) is a useful bacterium used to produce bacteriocin that can inhibit the growth of spoilage bacteria like Escherichia coli and Salmonella.

Reference

1.      http://en.wikipedia.org/wiki/Bacteriocin

2.      http://en.wikipedia.org/wiki/Agar_diffusion_test

3.      http://en.wikipedia.org/wiki/OD

4.      http://en.wikipedia.org/wiki/Zone_of_inhibition

5.      http://en.wikipedia.org/wiki/Absorbance