modern bioprocess technologist

Like other applications of biotechnology, modern bioprocess technology is an extension of ancient techniques for developing useful products by taking advantage of natural biological activities. When our early ancestors made alcoholic beverages, they used a bioprocess: the combination of yeast cells and nutrients (cereal grains) formed a fermentation system in which the organisms consumed the nutrients for their own growth and produced by-products (alcohol and carbon dioxide gas) that helped to make the beverage.
Although more sophisticated, today's bioprocess technology is based on the same principle: combining living matter (whole organisms or enzymes) with nutrients under the conditions necessary to make the desired end product.

Tuesday 5 April 2011

Lab6: Extraction of Plasmid DNA using GF-1 Plasmid DNA extraction Kit

Introduction

          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. This it uses a specially-treated glass filter membrane fixed into a column to efficiently bind DNA in  the presence of high salt. Combining alkaline  lysis-SDS and  minicolumn spin technology, up o 20μg of plasmid DNA  from bacterial cultures can be isolated.This 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 of DNA onto  a specially-treated  glass filter  membrane for efficient  recovery of highly pure plasmid DNA.

          The purification is based on the usage of denaturing agents to provide efficient cell lysis, denaturation of proteins and subsequent release of DNA. Special buffers provided in the kit are optimized to enhance binding of DNA onto a specially-treated glass filter membrane for efficient recovery of highly pure plasmid DNA
Plasmids are double-stranded circular DNA molecules that have the property of self-replication, independent of chromosomal DNA. Although the presence of a plasmid in a bacterial cell may be detected genetically as a change in phenotype, often it is necessary to isolate plasmid DNA for molecular studies, such as size determination, restriction enzyme mapping, and nucleotide sequencing, or for the construction of new hybrid plasmids. The degree of purification required will depend upon the intended use.

          In the process, the modified alkaline lysis method and RNase treatment are used to get cleared cell lysate plasmid DNA in crude lysate has been bound to the column; the contaminants can be washed off with wash buffer. Finally, the purified plasmid DNA is eluted by a high salt buffer and then precipitated with isopropanol for desalting. The entire procedure can be completed in 120minutes without ultracentrifuges and HPLC or other toxic reagents.

Storage Conditions
Plasmid DNA Extraction Kit can be stored at room temperature (15-25 ). After adding RNase A, PDE Buffer1, should be stored at 4 and is stable for six months. Other buffers and columns can be stored dry for up to 1 year at room temperature (15-25 )

Chemical Hazard
Buffer NB contains guanidine salts which can be harmful when in contact with skin orswallowed.
Always wear gloves and practice standard safety precautions. Do NOT disinfect guanidine orextraction waste in solutions containing bleach or any other form of acid. To clean any itemscontaminated with the reagent, simply soak in detergent and water to remove all traces ofguanidine before cleaning with bleach or acidic solutions.









Result

OD230 = 0.229
OD260 = 0.371
OD280 = 0.178

Ratio (OD260/ OD280)
= (0.371 ÷ 0.178)
= 2.084

Ratio (OD260/ OD230)
= (0.371 ÷ 0.229)
= 1.620

OD260 = 0.371
DNA concentration (µg/mL)
= 50 µg/mL x OD260 x dilution factor
= 50 x 0.371 x 50
= 927.5µg/mL

Total yield in 500µL sample
= DNA concentration x volume of sample in milliliters
= 927.5 µg/mL x 0.50 mL
= 463.75 µg


Discussion

            First, the S1 added to the pellet and the cell is re-suspended completely by vortexing or pipetting. The cells are re-suspended completely because lysis will not occur if clumps of bacteria remain following an inefficient re-suspension procedure. Then, the S2 added. It mixed gently by inverting tube several times (4-6 times). Vortex could sheared the genomic DNA and leads to contamination. So, it been avoided.  Then, the tube incubated in ice for less than 5 min. It reduced non-supercoiled plasmid contamination in bacteria strains and precipitation could be carrying out more effective in the cold. Buffer NB added and mixed it gently by inverting until white precipitate form and centrifuged it. After centrifugation, the compact white precipitate been spun down and was separate from the supernatant. The supernatant was transfer into column assembled in a clean collection tube. In order to obtain maximum yield, the orientation of the column was fix during centrifugation at all the time. The column that has a triangle mark on the edge was place at a fixed position during centrifugation. After that, the column is washed with wash buffer, centrifuged and discarded the flow through. The column was centrifuge again to remove residual ethanol because the quality of DNA could be affect by ethanol. Subsequently, the enzymatic reactions are inhibited. Then, the column was place into a clean microcentrifuge tube.50 µL of Elution Buffer is added onto column membrane and stood for one minute and centrifuged it. The elution buffer is dispensed directly onto the centre of the membrane for complete elution. 490 µL of sterile water is added to dilute the mixture. Finally, the mixture was transfer in a cuvette. The absorbance at 230nm, 260nm and 280nm are measured.

            The ratio of OD260 to OD280 that we got was 2.084 that were approaching 1.80. This shown there had less contaminant with protein. If there was contaminant with protein, the OD260/OD280 will be significant less.

            Besides, the ratio of OD260/OD230 that we got was 1.620, which was greater than 1.50. This reading represent the present of organic compound or chaotropic salts in the sample. This shown the amount of salt present in the our sample were low because the greater the amount of salt, the lower the ratio.
The cells are lysed under very carefully controlled conditions, the DNA fragment are still very large , much larger than plasmid and could be removed with cell debris by centrifugation.

           The methods used to purify the plasmid DNA from the cell lysate rely on the small size and abundance of the plasmid DNA relative to the chromosome, and the covalently closed circular nature of plasmid DNA. Most plasmids existed in the cytoplasm of the cell as circular DNA molecules that are highly supercoiled.

            Note that by not adhering to the recommended protocols, unsatisfactory results related to yield and quality of DNA may occur. The table below is problems that may occur and ways to overcome it.

Problem
Possibility
Suggestions
Low DNA yield
Cell lysis incomplete /
Lysate did not clear after
addition of S2
Do not exceed the recommended
culture volume of 5ml


Use fresh S2 by preparing as follows: 0.2N NaOH, 1% SDS.

Poor resuspension of cells
Ensure that cells are completely
resuspended after the addition of
S1. No cell clumps should be
visible

Low copy-number plasmid
Increase culture volume or grow culture in enriched medium such as Terrific Broth to increase the yield.

Bacteria culture overgrown
or not fresh
Do not culture bacteria longer than 20 hours at 37°C as this may lower the plasmid yield. Media should contain antibiotic at an appropriate concentration.

Column not placed at fixed
orientation during
centrifugation
Place the column which has a triangle mark on the edge, at a fixed position during centrifugation at all times.

Elution is not performed
properly
Ensure that the elution buffer used is a low salt buffer or water with a pH range of 7.0 - 8.5.
Column clogged
Transfer of precipitate from
sample prior to loading into
column
Ensure that white precipitate is not transferred over during loading of column to prevent clogging up of the membrane.
High molecular weight DNA contamination.
Vigorous mixing of lysate
upon addition of S2 or
Buffer NB
Do not vortex or mix vigorously after addition of S2 or Buffer NB. Simply mix by gently inverting the tube a few times.

Incubation longer than 5
min after addition of S
Do not incubate longer than 5 minutes.
Additional plasmid
formation
Irreversible denaturation
during cell lysis
Do not carry out incubation longer than 5 minutes after the addition of S2.

Nicked circular plasmids
due to the presence of
nuclease
Carry out purification without delay at least until the washing step where nucleases will be removed.


Incubation on ice after addition of S2 reduces nuclease activity.
RNA Contamination
RNA digestion was
insufficient
Ensure that RNase A has been added into S1 or add a new preparation of RNase A into S1 to a final concentration of 100µg/ml.
Poor performance of
eluted DNA in
downstream
applications
Eluted DNA contains traces
of ethanol
Ensure that the Column drying step carried out prior to elution.


Use Elution Buffer or water with a pH range of 7.0 - 8.5.

Conclusion

          Extraction is an easy and quick way to purify DNA from a mixture of proteins, lipids and nucleic acids (e.g., a cell or bacterial lysate). The extract is deprotenised, the RNA be removed and the DNA is concentrated by ethanol precipitation. In a plasmid preparation, the plasmid DNA from the large amount of bacteria chromosomal DNA is necessary to be separate.

          Plasmid DNA Extraction Kit was design for rapid and efficient purification of high copy and low copy plasmid DNA without the need for precipitation or organic extractions. It used a specially treated glass filter membrane fixed into a column to bind DNA efficiently in the presence of high salt. Special buffers provided in the kit were optimizing to enhance binding of DNA onto a specially treated glass filter membrane for efficient recovery of highly pure plasmid DNA. Enzymes added to purify DNA in vitro could have unhindered access to it. These enzymes could be use for restriction mapping, ligation, sequencing, or other procedures to modify the DNA.
         Multiple samples could be process rapidly and with practice, the purification takes less than 30 minutes. Optimized buffers ensure only highly pure plasmid DNA is extracted. It was ready to use in many routine molecular biology applications such as restriction enzyme digestion, radioactive/fluorescence DNA sequencing, PCR, ligation, transformation and other manipulations.



Reference
Book:
Biotechnology-4 Including recombinant DNA technology,EnvironmentalBiotechnolog,Animal cell culture by S.Mahesh and A.B Vedamurthy
Bacterial Genetics by Nancy Trun and Janine Trempy





Lab 5: Determination of Antimicrobial Effects of Microbial Extracts

Introduction   
An anti-microbial is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans. Antimicrobial drugs either kill microbes (microbiocidal) or prevent the growth of microbes (microbiostatic). Disinfectants are antimicrobial substances used on non-living objects or outside the body. Antibiotics are the thing that inhibit the growth of pathogenic and spoilage microorganism. Organic acids and bacteriocins are included among this antimicrobial compound.
Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They are typically considered to be narrow spectrum antibiotics, though this has been debated.[1] They are phenomenological analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse.
The lactic acid bacteria (LAB) comprise a clade of Gram-positive, low-GC, acid-tolerant, generally non-sporulation, non-respiring rod or cocci that are associated by their common metabolic and physiological characteristics.  Proteinaceous bacteriocins are produced by several LAB strains and provide an additional hurdle for spoilage and pathogenic microorganisms. The range of inhibitory activity by these bacteriocins of lactic acid bacteria can be either narrows, inhibiting only those strains that are closely related to the producer organism or wide, inhibiting a diverse group of Gram-positive microorganisms. Thebacteriocins have been reported to inhibit the growth of bacteria; in this experiment we use them to inhibit growing of Staphylococcus aureus and Escherichia coli.
Staphylococcus aureus is a facultative anaerobic, Gram-positive coccus, and is the most common cause of staph infections. It is frequently part of the skin flora found in the nose and on skin. S. aureus can cause a range of illnesses from minor skin infections, such as pimple, impetigo, boils (furuncles).  It is still one of the five most common causes of nosocomial infections, often causing postsurgical wound infections.
Escherichia coli is a Gram-negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some can cause serious food poisoning in humans. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2 and by preventing the establishment of pathogenic bacteria within the intestine.

Part I: Determination of bacteriocin activity via agar diffusion test
The agar diffusion test, or the Kirby-Bauer disk-diffusion method, is a means of measuring the effect of an antimicrobial agent against bacteria grown in culture.
The bacteria in question are swabbed uniformly across a culture plate. A filter-paper disk, impregnated with the compound to be tested, is then placed on the surface of the agar. The compound diffuses from the filter paper into the agar. The concentration of the compound will be highest next to the disk, and will decrease as distance from the disk increases. If the compound is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is thezone of inhibition. Thus, the size of the zone of inhibition is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound.

Part II: Determination of bacteriocin activity via optical density
Optical density, measured in a spectrophotometer, can be used as a measure of the concentration of bacteria in a suspension. As visible light passes through a cell suspension the light is scattered. Greater scatter indicates that more bacteria or other material is present. The amount of light scatter can be measured in a spectrophotometer. Typically, when working with a particular type of cell, you would determine the optical density at a particular wavelength that correlates with the different phases of bacterial growth. Generally we will want to use cells that are in their mid-log phase of growth. Typically the OD600 is measured.

Objective
Refer to the lab manual

Material and reagents
Refer to the lab manual

Procedure (Part I: Determination of bacteriocin activity via agar diffusion test)
Refer to the lab manual

Material and reagents
Refer to the lab manual

Procedure (Part I: Determination of bacteriocin activity via agar diffusion test)
Refer to the lab manual

Result (part 1)
Calculations 
Inhibition zone :
Serial dilutions of extracellular extract

Y axis : Abs600  or OD600                                                                         X axis : 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 . Therefore , x = ( Y – c)/m
When Y = Z/2 , thus, x= (Z /2-c)/m

Data sheet
Part I. Determination of bacteriocin activity via agar diffusion test.

Strains of LAB
Strains of spoilage/pathogenic bacteria
Inhibition zone (cm )
LAB species
Staphylococcus aureus
(0.85+0.80)/2=0.825
Escherichia coli
(0.70+0.70)/2=0.70

Discussion (part 1)

Lactic acid bacteria produce a variety of antagonistic factors that include metabolic end products, antibiotic-like substances and bactericidal proteins, termed bacteriocins. The range of inhibitory activity by bacteriocins of lactic acid bacteria can be either narrow inhibiting only those strains that are closely related to the producer organism, or wide, inhibiting a diverse group of Gram-positive microorganisms.

The extracts of the lactic acid bacteria gave zones of inhibition onto the indicator pathogen strains tested. In the agar well diffusion assay a linear relationship existed between response (diameter or area of the zone of inhibition)The strains inhibited were Escherichia coli and Staphylococcus aureus. The diameter of inhibition zone obtained from Escherichia coliwas0.700cm while the diameter of inhibition zone obtained from Staphylococcus aureuswas 0.825cm.Staphylococcus aureus was a gram positive bacteria while Escherichia coli were gram negative bacteria. The result showed us that the diameter of inhibited zone in Staphylococcus aureus was larger than Escherichia coli. This was because a gram positive indicator bacterium was much more sensitive to bacteriocin of lactic acid bacteria strains than gram negative indicator bacteria. The resistance of gram negative bacteria was attributed to the particular nature of their cellular envelope, the mechanisms of action described for bacteriocin bringing in a phenomenon of adsorption.
According to Bhunia et al. (1991) the pediocin(bacteriocin produced by  Pediococcusacidilactici) interacts with lipoteichoic acids absent in gram negative bacteria. These molecules play the role of site of necessary not specific reception to produce the bactericidal effect. Some bacteriocins produced by gram positive bacteria had a broad spectrum of activity. These variations of sensibility were due to the characteristic of indicators strains and thus in level of hurt caused by the inhibitive factor.



Procedure (Part II: Determination of bacteriocin activity via optical density)
Refer to the lab manual


Result (part2)

Part II. Determination of bacteriocin activity via optical density
Serial dilution of extracellular extract
Dilutions
OD600 of spoilage/pathogenic bacteria
Staphylococcus aureus
Escherichia coli
0X
0.304
0.623
2X
1.087
2.044
10X
1.207
2.283
50X
1.022
2.525
100X
1.794
2.012
Equation
y = 0.3259x + 0.9197
y = 0.2915x + 0.2083
OD600  of control
0.322
0.322
50% of OD600
0.161
0.161
AU/ml
-2.33
-0.162

Staphylococcus aureus

 
Escherichia coli


Discussion (Part II)
            The activity of bacteriocins was difficult to quantify and was dependent on the determination method. The most widely used techniques were based on the evaluation of growth inhibition caused on a sensitive bacterial strain, either in solution or in agar plates. A standardized bioassay does not exist because bacteriocinswere significantly different among them, making difficult the use of a common standard, and results depend on experimental conditions. The advantages of using this method were the elimination of diffusion related problems, its quickness, commodity and low cost. Major experiment errors came from the cell sedimentation and interference of sample colour. In addition, time of reaction was generally critical and the relationship between the bacteriocin concentration and the inhibitory response usually follow a sigmoidal curve making use of complex regression models of limited practical application.The logarithm of the dose while a non-linear equation was used to model the sigmoidal dose/response curve in photometric assays (PA). The dose/response curves were used to define titters of the standard solutions in arbitrary units and to develop quantitative assays for all the bacteriocins.
Generally, the OD value is higher from the 100X dilution. It indicated that the LAB has stronger antimicrobial effect on the pathogenic bacteria compare to a more diluted solution. The OD600 values in 100X dilutions had the highest value in both pathogenic bacteria where Staphylococcosaurens is 1.794 and Escherichia coli is 2.012.So, the more diluted the extracellular extract, the greater the value of OD600. Both of the graphs of Staphylococcus aureusand Escherichia coli are obtained by plotting the optical density of spoilage/pathogenic bacteria Staphylococcus aurensor Escherichia coli (OD600) against different serial dilutions of extracellular extract. The graph demonstrated a less linear relationship between serial dilutions of extracellular extract and OD600, with the regression analysis giving an R2 value of 0.4825 for Staphylococcus aureus.While the graph of Escherichia colidemonstrated a more linear relationship between serial dilutions of extracellular extract and OD600, with the regression analysis giving an R2 value of 0.751.




Conclusion
In summary, the present results clearly suggest the potential usefulness of the bacteriocins produced by Lactic acid bacteria (LAB) as bio preservatives against both Escherichia coli and Staphylococcus aureus. Both acidification and the production of hydrogen peroxide by LAB were ruled out as the source of the inhibition. LAB competed with spoilage microorganisms, such as certain Gram-negative bacteria for nutrients or space with. Moreover, the shelf-life of food products could extend the production of organic acids, hydrogen peroxide, low molecular weight metabolites (such as diacetyl and bacteriocins) due to their inhibiting effect on the growth of spoilage and pathogenic bacteria. For these reasons, antagonistic effects against pathogenic bacteria exhibited by harmless LAB applied interest as bio preservatives – with the subsequent reduction in the use of antibiotics – in future aquaculture activities.
Bacteriocins have been defined as proteinaceous substances exhibiting bactericidal activity against closely related species. Currently they are receiving increased attention because of their inhibitory activity against food spoilage and food-borne pathogenic bacteria such as Listeria monocytogenes. Commercial nisin preparations have been evaluate in food systems. It was now widely used as bio preservatives in the food industry due to their antibacterial properties. This allowed a more strict microbial control of a variety of commercial food products.
Determination of bacteriocin activity by the agar diffusion assay in which inhibition zones are produced in plates in a procedure similar to that of antibiograms, is undoubtedly the most commonly used despite the inconveniences and limitations of its application. The performance of the method, which is laborious and time-consuming, depends largely on human ability and judgment and precision can’t be achieved when inhibition zones are unclear or not perfectly circular. Diffusion-related difficulties of the active substance represent another important limitation of agar diffusion assays. The need to eliminate diffusion-related problems associated with the agar techniques, introduced liquid medium methods, which make use of indicator organisms and quantify the bacteriocin concentration from the percentage of growth inhibition in the indicator organism. Since then, applications of turbidometric assays by spectrophotometer can be found in a number of reports in which, as with the agar diffusion assay, various indicator microorganisms were used, in procedures that show large variability regarding bacteriocin extraction, general experimental conditions and definition of the bacteriocin unit. Sensitivity limits and linearity of responses to various bacteriocin levels vary significantly among different test-microorganisms in both bioassays, the lower sensitivity limits depending on both the test-microorganism and the applied method. Very low nisin concentrations, e.g. 1 IU/ml, were more safely determined in the turbidometric assay (spectrophotometer) through determination of the percentage of inhibition of growth of the indicator microorganism. This method proved to be more suitable for determination of nisin in processed food samples.
Although the agar diffusion assay is the most widely used method in routine measurements of bacteriocin activity, turbidometry (spectrophotometer) offers a simpler, faster and more reliable alternative since diffusion related problems are eliminated, the degree of human intervention and judgment is low, and very low bacteriocin concentrations can be quantified.



Reference