LAB 6: Bacterial DNA Extraction

INTRODUCTION

            Plasmid is a small DNA molecule within a cell that is physically separated from a chromosomal DNA which can replicate independently. Plasmid are commonly found in bacteria as small circular, double stranded DNA molecule.

            Plasmid are involved in the DNA recombinant experiment to produce large quantity of DNA. Plasmid act as vector to clone specific gene in DNA.  They are often found in Gram-positive bacteria and Gram-negative bacteria.    

            The GF-1 Plasmid DNA Extraction Kit is designed for rapid and efficient purification of high copy and low copy plasmid DNA from bacterial lysates. The kit uses the alkaline lysis-SDS method to lyse cells and release plasmid DNA. Special buffers provided in the kit are optimized to enhance binding DNA onto a specially-treated glass filter membrane for efficient recovery of highly pure plasmid DNA. The kit will produce up to 20μg of DNA and produce the DNA rapidly. No organic-based extraction is needed. It also produce highly pure plasmid DNA which ready to use for routine molecular biology applications such as restriction enzyme digestion, PCR, ligation, DNA sequencing, transformation, etc.

            DNA purity can be determined by measuring the intensity of absorbance of the solution at wavelengths 260nm and 280nm.

OBJECTIVE

1.       To determine the method to extract the plasmid DNA from bacteria.

2.       To determine the difference of plasmid extraction between Gram-positive bacteria and Gram-negative bacteria.

MATERIALS AND REAGENTS

Centrifuge

Pipette

Incubator (Digital Dry Bath)

Column

Microcentrifugation tube

Buffer R1

Buffer R2

Proteinase K

Ethanol

Buffer BG

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.

·       Water bath was pre-set to 37°C and the second water bath to 65°C.

1. Centrifugation.

Pellet 1 ml of bacteria culture grown overnight or culture grown to log phase was centrifuged at 6,000 x g for 2 min at room temperature. The supernatant was decanted completely.

2. Re-suspension of pellet.

100µl Buffer R1 was added to the pellet and the cells were re-suspended completely by pipetting up and down or shake vigorously.

3. Lysozyme treatment.

For Gram-negative bacteria strains, 10µl lysozyme (50mg/ml) was added into the cell suspension. For Gram-positive bacteria strains,20µl lysozyme (50mg/ml) was added into the cell suspension. The mixtures were then mixed thoroughly and incubated at 37°C for 20 min.

4. Centrifugation.

Pellet was formed when digested cells were centrifuged at 10,000 x g for 3 min. The supernatant was decanted completely.

5. Protein denaturation.

Pellet was re-suspended in 180µl of Buffer R2 and 20µl of Proteinase K was added. The mixture was mixed thoroughly. They were then incubated at 65°C for 20 min in a shaking water bath or with occasional mixed every 5 min.

6. Homogenization.

2 volumes (~400µl without RNase A treatment, ~440µl with RNase A treatment) of Buffer BG was added and mixed thoroughly by inverting tube several times until a homogenous solution was obtained. It was incubated for 10 min at 65°C.

7. Addition of Ethanol.

200µl of absolute ethanol was added. The sample was mixed immediately and thoroughly.

8. Loading to column.

The sample (max. 650µl) was transferred into a column assembled in a clean collection tube (provided). It was then centrifuged at 10,000 x g for 1 min. Flow through was discarded. The remaining samples from step 7 were repeated the same step.

9. Column washing.

The column was washed with 650µl of Wash Buffer and centrifuged at 10,000 x g for 1 min. The flow through was discarded.

10. Column drying.

The column was centrifuged at 10,000 x g for 1 min. The residual ethanol was removed.

11. DNA elution.

The column was placed into a clean microcentrifugation tube. 50 µl of preheated Elution Buffer was added, TE buffer or sterile water directly onto column membrane and stand for 2 min. DNA was eluted by centrifuged it at 10,000 x g for 1 min. The DNA was then stored at 4°C or -20°C.

RESULT

	Gram Positive Bacteria	Gram Negative Bacteria
OD230	2.363	3.375
OD260	0.688	0.600
OD280	0.346	0.313
Ratio (OD260/OD280)	1.99	1.92
DNA Concentration (µg/mL)	674.509	588.240

DISCUSSION

1.       The bacterial has to be grown overnight to ensure that the bacterial is all grown to the phase needed.

2.     10µlof Lysozyme is added to the Gram Negative bacteria-strain while 20µl of lysozyme is added to the Gram Positive bacteria-strain because the Lysozyme is first needed to change the Gram Positive strain to Gram Negative and then deactivate both of it .

3.       During the extraction of DNA, there is a lot of contaminating proteins present. These contaminants must be removed. Proteinase K is used to digest these contaminating proteins. In addition, there may be enzymes that degrade nucleic acid present, proteinase K degrades these enzymes and protects the nucleic acids from attack. In addition, proteinase K is stable over a wide pH range and is well suited for use in DNA extraction.

4.       Ethanol is used in DNA extraction to force the DNA to precipitate in a solution. In order to collect a DNA sample, cells are broken down through agitation, then mixed with ethanol to create an aqueous solution. Ethanol works to prevent the DNA from dissolving into the water, instead causing it to precipitate out so it can be separated and extracted using a centrifuge.

5.       Ratio of OD260/OD280 is to to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. In this case, the ratio of both Gram Positive and Gram Negative Bacteria show ratio of 1.99 and 1.92 respectively indicates that the it shows the pure DNA.

6.       The DNA concentration is calculated by using Beer Lambert’s equation which is

            AxBxC whereby

            A: the wavelength-dependent absorptivity coefficient of OD260

            B: path length = 0.051 ml/µm

            C: analyte concentration = 0.02

CONCLUSION

Extraction of DNA is now becoming very important in research field as more product is coming out from the DNA especially human DNA by replicating it. In this case a pure and clean DNA has to be produce. And thus, the precautions have to be take noted and contamination of DNA must be avoided in order to produce pure DNA. The method of isolation of plasmid DNA can successfully extract the DNA out. The concentration of DNA extracted from gram positive bacteria is higher than the one from gram negative bacteria because the alkaline lysis is more effective towards the gram positive bacteria.

Lab 5: Determination of antimicrobial effects of microbial extracts

Introduction
Human being nowadays are more concern about the naturality of the product and try to consume natural made but not artificial made product, thus antimicrobial substance are now widely investigate and used in the daily products especially food.

Antimicrobial is any substance of natural, semisynthetic or synthetic origin that kills or inhibits the growth of microorganisms but causes little or no damage to the host. It is produced by bacteria and one of its example is bacteriocins that widely used in food industry. The bacteria that we used in this experiment is LAB1 and LAB2 to produce bnjacteriocins as its extracellular extracts. Bacteriocins are proteins or complexed proteins biologically active with antimicrobial action against pathogen. They are normally not termed antibiotics in order to avoid confusion and concern with therapeutic antibiotics, which can potentially illicit allergic reactions in humans and other medical problems. 

Objective
To determine the antimicrobial effects of extracellular 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
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

Procedure
Part I.   Determination of bacteriocin activity via agar diffusion test
1. All the petri dishes were labelled according the spoilage organisms and strains of LAB used
2. Each plate was used for one strain of spoilage organism and one strain of LAB. The plate was divided into 2, each side for one replicate.
3. Each group was given 2 strains of LAB and 2 strains of pathogenic organisms.
4. 10 ml of trypticase soy-yeast extract agar (TSAYE) was loaded into the labelled petri dishes and ensure that the agar covers the entire surface of the plate. Wait for it to solidify.
5. 2 ml of the broth containing the spoilage organism was inoculated into 10 ml of Brain Heart Infusion (BHI) agar and vortex.
6. The mixture was loaded on top of the TSAYE agar layer, ensure that it covers the entire surface, and wait for it to solidify.
7. The broth containing LAB cultures was centrifuged. The supernatant was used as extracellular extracts.
8. A sterile filter paper disk was aseptically picked up with sterile forceps and  the disk was dipped into the extracellular extract. (Be sure that the excess extract has drained off).
9. The paper disk was placed on top of the solidified BHI agar.
10. The plates were incubated for 24-48 h at 37 °C.
11. Upon incubation, the inhibition zones were measured (in cm) and the reading was recorded in the table 1.

Part II. Determination of bacteriocin activity via optical density
1. The broth containing LAB cultures was centrifuged. The supernatant was used as extracellular extracts.
2. Each group was given 2 strains of LAB and 2 strains of pathogenic organisms.
3. 5 ml of double-strength MRS was added with 1 ml of cultures containing pathogenic bacteria and the mixture was vortexed.
4. A serial dilution of the extracellular extracts (diluted 0x, 2x, 10x, 50x, 100x) was prepared.
5. 5 ml of each extracellular extracts dilution was added into mixture as prepared in step (3).
6. The mixtures were incubated for 12-15 hr at 37 °C.
7. A control using 10 ml of double-strength MRS and 1 ml of cultures containing pathogenic bacteria was prepared and the mixtures was incubated for 12-15 hr at 37 °C as well.
8. Upon incubation, measure the optical density of the spoilage/pathogenic bacteria at 600 nm. Perform the same for the control as well.
9. 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.
10. 50% of the spoilage/pathogenic bacteria growth were determined from the OD600 of the control.

Calculations
Inhibition zone:
 Get the average of the 2 inhibition zone.

Arbitrary unit:
y:   Abs600 or OD600                
x:   Serial dilutions of extracellular extract 
m and c:   Constants

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

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

Result 
Part I  Determination of bacteriocin activity via agar diffusion test

Figure 1 shows samples of LAB 1 with Escherichia coli 

Figure 2 shows samples of LAB 1 with Salmonella

Figure 3 shows samples of LAB 2 with Escherichia coli 

Figure 4 shows the samples of LAB 2 with Salmonella

Strains of LAB	Strains of spoilage/pathogenic bacteria	Inhibition zone (cm)		Average (cm)
LAB 1	Escherichia coli	0.8	1.0	0.9
	Salmonella	1.1	0.9	1.0
LAB 2	Escherichia coli	0	0	0
	Salmonella	1.3	1.1	1.2

Table 1: The Result of Determination of Bacteriocin Activity via Agar Diffusion Test














Part II.   Determination of bacteriocin activity via optical density

Serial dilution of extracellular extract

Strain of LAB 2

Dilutions	OD600 of pathogenic bacteria
	Strain 1: E. coli	Strain 2: Salmonella
0x	0.219	0.218
2x	0.586	0.657
10x	0.927	0.764
50x	1.035	0.750
100x	0.968	0.734
Equation	y = 0.1947x + 0.1629	y = 0.1125x + 0.2871
OD600 of control	0.05	0.054
50% of OD600	0.067	0.051
AU/ml	-0.4926	-2.0987

Table 2: OD600 of pathogenic bacteria with different dilution

Graph 1: Graph of Abs600 of E. coli versus serial dilution of extracellular extract.

Graph 2: Graph of Abs 600 of Salmonella versus serial dilution of extracellular extract

Discussion :
Part I  Determination of bacteriocin activity via agar diffusion test

1. Lactic acid bacteria (LAB) is a clade of Gram-positive, low-GC, acid-tolerant, generally non-sporulating, non-respiring, either rod-shaped or coccus-shaped bacteria that share common metabolic and physiological characteristics. LAB produce bacteriocin which are proteinaceous toxins that inhibit the growth of its similar or closely related bacterial strain.
2. Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms. Salmonella species are Gram-negative, flagellated facultatively anaerobic bacilli characterized by O, H, and Vi antigens.
3. The agar diffusion test is used to measure the effect of LAB against the growth of Escherichia coli and Salmonella in the culture. The two bacteria are swabbed uniformly on the culture plate respectively. The filter paper disks are dipped with one of the LAB culture is then placed on the surface of agar. The LAB diffuse into the agar from the filter paper. If the LAB is effective against the bacteria on the agar, zone of inhibition will present. It is the clear region that can found around the filter paper on the agar. The diameter of the zone of inhibition is a measure of the effectiveness of LAB. The larger the diameter of zone inhibition, the greater the effectiveness of bacteriocin in LAB. 
4. The average diameter of zone of inhibition obtained from the Escherichia coli is (0.9+0)/2=0.45cm while the average diameter of zone of inhibition obtained from the Salmonella is (1.0+1.2)/2=1.1cm. Since Salmonella has the greater diameter of inhibition zone, the effectiveness of bacteriocin is greater.

Part 2: Determination of bacteriocin activity via optical density

1. Optical density measurement of bacterial culture is a common technique used in microbiology. Spectrophotometer is an instrument used to measure Optical density. It also can be used to measure the concentration of bacteria in a suspension. When visible light passes through a cell suspension, the light is scattered. Spectrophotometer can be measured the amount of light scattered by the cell suspension. The greater scatter indicates that the presence of more bacteria or other material. Typically, the optical density at a particular wavelength that correlates with the different phases of bacterial growth can be determined when working with a particular type of cell. Generally, the cells that are in their mid-log phase of growth are being used. Typically the OD600 is measured. 

2. Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution.The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. The amount of a known chemical substance can be measure by measuring the intensity of light detected. Spectrometer is a term that is applied to instruments that operate over a very wide range of wavelengths, from gamma rays and X-rays into the far infrared. If the instrument is designed to measure the spectrum in absolute units rather than relative units, then it is typically called a spectrophotometer. According Beer-Lambert Law, amount of light absorbed by a medium is proportional to the concentration of the absorbing material or solute present. In this case, the concentration of a coloured solute in a solution could be determined in the lab by measuring the absorbency of light at a given wavelength.

3. Arbitrary unit (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.
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
4. From the result obtained from the experiment, Escherichia coli and Salmonella shows a negative gradient graph. This shows that there has a  negative inhibition from the bacteriocin produced by lactic acid bacteria (LAB) on the growth of the bacteria strains. 

Conclusion:

The results show that lactic acid bacteria

(LAB) may act as a bio preservatives. Antimicrobial compounds produced by LAB have provided these organisms with a competitive advantage over other resistance to heat, acidity, low water activity and oscillations of microorganisms. In addition, these molecules present characteristics of effective natural inhibitors of pathogenic and food spoilage bacteria in temperature. Lactic acid bacteria produced inhibitory substances are safe and various food.