Modern Biotechnology 2011 (Synthetic biology) - Science of the Unthinkable.!!

Three commonly used affinity tags for protein purification

In order to purify heterologous proteins from various hosts, affinity tags are extremely capable tools. In the recent years affinity tags have become highly popular tools for protein purification mainly due to following reasons. They provide high level of purification of recombinant proteins from crude extracts, mostly in a single step. Second, they provide mild elution conditions, thereby, do not interfere with the structure and hence the function of the purified proteins. In addition, affinity tags allow a variety of proteins to be purified using easy procedures.

Affinity tags are available as expression vector systems having multiple cloning sites (MCS) for cloning the gene of interest towards the N or C-terminal of the tag. Now-a-days, a variety of affinity tags are available for the purification of recombinant proteins. Each tag has its certain advantages and disadvantages. Here, you will see the properties of the three commonly used affinity tags: His-Tag, GST-Tag and MBP-Tag.

1)  His-Tag

His-tag is the most commonly used affinity tag for the purification of recombinant proteins in E.coli. The His-tag is a peptide motif that consists of six histidine (His) residues. Therefore, it is also called as hexa histidine-tag or 6xHis-tag. The most commonly used bacterial expression vector system for His-tag is the pET series (Novagen). These vectors provide both the N & C-terminal fusion of gene of interest with the His-tag.

It is observed that hexahistidine has high affinity towards transition metals e.g. Ni⁺⁺, Co⁺⁺ etc. Therefore, in order to purify recombinant proteins with hexa histidine-tag, immobilized-metal affinity chromatography (IMAC) columns are used. Ni-NTA agarose is the most commonly used resin for the purification of His-tag proteins. Since very few naturally occurring proteins bind to the Ni-NTA matrices with considerable affinities, therefore, recombinant proteins containing the His-Tag are significantly purified in a single step.

The recombinant protein is generally eluted either with the lowering of pH or with the addition of imidiazole to the column.

Advantages:

• Hexahistidine tag is much smaller and hence provides high yields of tagged proteins.
• It does not interfere with the structure and function of the recombinant protein.
• Its affinity for Ni-NTA matrix is non-dependant on the conformation of the target protein.
• The elution conditions for His-tagged protein are milder.
• Ionic strength, chaotropic agents and detergents do not affect the purification of His-tag proteins.
• Less expensive.

Because of all these advantages, His-Tag is the affinity tag of choice for various protein expression and purification experiments.

The only disadvantage of the tag is that certain bacterial proteins are able to bind to the His-Tag and hence are co-eluted with the recombinant protein. This can be partially ruled out by increasing the stringency of washing.

2)  GST-Tag

The Glutathione S-transferases (GSTs) are class of enzymes that are involved in cellular defense against small toxic compounds. They are abundant enzymes that utilize glutathione as a substrate. GSTs bind to glutathione with high affinity and specificity. Therefore, they provide an efficient system for the purification of proteins.

Generally, the Glutathione S-transferase (GST) from Schistosoma japonicum is used as the affinity tag in the pGEX vector series. The gene encodes a protein of 218 residues having molecular weight of 26KDa. The fusion protein thus produced can be purified using the glutathione-based affinity resins. The strength and selectivity of the resin for GST-tagged proteins results in the successful purification of the recombinant protein from the cell extract, in a single step.

In order to elute the recombinant proteins, reduced glutathione is added to the column. This allows the elution of recombinant proteins under mild and non-denaturing conditions.

Advantages:

• Provides a higher degree of purification in a single chromatographic step.
• Increases the solubility of the recombinant protein.
• It does not interfere with the structure and function of the recombinant protein.
• Provides immunogenic as well as biochemical assay of the recombinant protein.
• The elution conditions for GST-tagged protein is milder than most of the affinity purification methods.

Disadvantages:

• Due to its larger size it is prone to degradation by proteases.
• Affinity for glutathione resin depends on certain reagents.
• More expensive.
• In order to study the protein of interest in detail the tag has to be removed.

Nonetheless, GST-tag is a versatile tag for protein expression and purification. It is generally used when protein of interest is not expressed in His-tag system or is having solubility or purity issues.

3)  MBP-Tag

Maltose Binding Protein (MBP) is a periplasmic protein of the bacterium E.coli. The protein is a component of the maltose and maltodextrins. It is encoded by the gene malE. The malE gene product bears a wide variety of fusions and hence is suitable for expressing proteins which do have problems in expression, in His-Tag or GST-Tag systems.

The expression vector system consisting of MBP as the affinity tag is pMAL series (New England Biolabs).

As MBP is a component of maltose, therefore, the fusion protein can be purified using matrices consisting of sugars. Generally amylose resins are used for the purification of MBP tagged proteins. For the elution of the recombinant protein maltose is added to the column. As a result of this, the elution of recombinant proteins is mild and under non-denaturing conditions.

Advantages:

• Provides a higher degree of purification in a single chromatographic step.
• Increases the expression as well as solubility of the recombinant protein.
• It does not interfere with the structure and function of the recombinant protein.
• Allows periplasmic expression of the recombinant protein.
• Allows the formation of disulfide bonds in the foreign proteins.
• Limits degradation of the recombinant protein.
• Less expensive.

Disadvantages:

• Due to its larger size, expression of large proteins is sometimes problematic .
• In order to study the protein of interest in detail the MBP tag has to be removed

The elution conditions for MBP-tagged protein is milder than most of the affinity purification methods. In addition, due to bacterial origin of malE gene, MBP is a better tag in terms of expression and solubility as compared to GST.

These were the overview of the three most commonly used  affinity tags for the purification of heterologous proteins in E.coli. According to our experiences, one should start with the His-Tag, followed by GST and MBP. In most of the cases, His-Tag results in decent level of expression and purification of recombinant proteins. For any query or suggestions related to protein purification in E.coli, please feel free to post it in the comments.

Kuby Immunology 4th edition by Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne

Kuby Immunology is one of the top immunology textbooks on the academic market. Currently in its 6th edition, Kuby Immunology is written by a staff of top instructors in immunology.  The textbook is named after Janis Kuby, a former professor at San Francisco State University and the writer of the original edition.  It was originally produced as a textbook entitled Immunology, but became so well regarded in the field that it became officially designated as Kuby Immunology.  Kuby Immunology is, by and large, designed as an introductory textbook, and is thus used primarily on an undergraduate level in Pre-Med and Biology programs.  Kuby Immunology is highly regarded for being consistently comprehensive and up-to-date with many of the most crucial discoveries in the every changing and developing science of immunology.  Since it is designed as introductory, Kuby Immunology writes out many of its concepts in straight, easy to understand terms, largely avoiding more complicated academic language.  It covers most of the various  categories of immunology, and focuses greatly on the multi-disciplinary nature of immunology since much of its audience does not necessarily pursue careers in immunology.  For them, Kuby Immunology is the main text that they will ever read about immunology. 

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Encyclopedia of Microbiology

Humans have always struggled with how to balance the benefits bacteria offer with the threats that they produce. Much less obvious than the effects microorganisms have on plants and animals are the indirect ways in which they shape the planet. These hidden activities have rarely been explained in science, though scientists realize that the behavior of microbes supports all life on Earth.

In more than 200 entries, Encyclopedia of Microbiology presents the myriad ways in which microorganisms influence the biosphere. Focusing on how all microorganisms relate to each other just as all higher organisms relate to all other animate and inanimate objects on Earth, this new resource explores all aspects of microbiology from mycology (the study of fungi) to the simplest biological entities of all, viruses, to prions, which are even more streamlined than viruses and just as dangerous. Biographical sections in many entries highlight the scientists who most influenced developments or discoveries in microbiology, including Louis Pasteur. Entries cover new techniques in microscopy, genetic engineering, gene therapy, and nanotechnology. A full-color insert, helpful appendixes, cross-references, and further resources round out this invaluable resource.

Essays include:

• Where Are Germs Found?
• The Realities of Biological Weapons
• Does Immigration Lead to Increased Incidence of Disease?
• Will Global Warming Influence Emerging Diseases?
• Microbes Meeting the Need for New Energy Sources
• Bioengineered Microbes in the Environment
• Does Vaccination Endanger or Improve Our Health?
• Does Air Travel Make Us Sick?

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5 Factors affecting gene cloning efficiency

Gene cloning is the central technique involved in recombinant DNA technology. Moreover, it facilitates the discoveries and understandings of the gene structure, function and regulation. A new era has been initiated as a result of this method in the manipulation, analysis and exploitation of bio-molecules.

However, competent gene cloning is not that much easy as it sounds. In order to efficiently clone the gene of interest into a particular vector, you need to be skilled. Since the efficiency of cloning is determined by several factors. Therefore, each factor should be deliberately considered to get best cloning efficiencies.

Here we provide you the most common factors which affect the efficiency of your gene cloning experiment.

1)         Starting Material

Well begun is half done. You know this notion very well. This applies to gene cloning also. A good starting material means you have done half of the things correct, only remaining half is to be optimized.

You materials should be pure. The isolated plasmid should be free from contaminating components. One of the most common contaminant is the media in which the culture is grown. This results in the poor digestion and ligation of the plasmid.

Therefore, during plasmid isolation, you should make sure that the media has been completely removed from the bacterial cells. Moreover, it is also a good practice to ethanol precipitate the plasmids prior to restriction digestion. This removes the salts present in the plasmid suspension and hence results in proper digestion of the plasmids.

If PCR product is the starting material then either reaction cleanup or gel extraction should be performed. We generally go for the gel elution of the PCR product as this not only removes the reaction components and primer dimers but also the non-specific amplifications.

2)         Digestion of the Vector and Insert

Digestion is very important factor determining the efficiency of the cloning experiment. Good cloning efficiency requires complete digestion of the insert and vector molecules. Digestion, if not properly done results in partial digestion of the vector and insert molecules, which in turn results in poor cloning efficiency.

However, it is relatively uncomplicated to achieve complete digestion. You should follow the stuffs quoted here in order to get desired results.

You should take units of the restriction enzyme according to the amount of plasmid or PCR fragment taken. Generally, 1-2µg of plasmid DNA/PCR product is a good amount for digestion. To digest this amount of DNA you should take 1-2µl (5-20 units) of the chosen restriction enzyme. Now-a-days many suppliers provide enzymes in a format that contains units in 1µl to digest 1µg of DNA. This really simplifies the digestion process.

Things become more complicated when you have to perform double digestion. While performing double digestion you should always check the buffer in which both the enzymes are having maximum activity. But there are certain enzymes which cannot be used for double digestion. The reason being the fact that there buffer requirements are not compatible. In that case it is better to perform sequential digestion.

However, sequential digestion results in the loss of the fragment so you will have to start with more amount of DNA. In addition, there are certain enzymes which have different optimum temperatures. In this case also you will have to go for sequential digestion.

However, there are certain suppliers (NEB, Fermentas etc) who have optimized a single buffer and a single temperature for a variety of commonly used enzymes. Unfortunately, these enzymes are little bit expensive but in our opinion are worth. Using such enzymes not only saves your time and energy but also improves the digestion many folds.

3)         Amount of digested vector to be taken for ligation

Well, after completely digesting the insert and the vector molecules, the question arises about the quantity of digested vector to be used for ligation. This significantly affects the efficiency of your gene cloning experiment.

The amount of vector to be taken solely depends on the size of the vector. For example, if the size of the vector is small then you will have to take little amount of vector and vice-versa.

We have made a generalized approach towards the size and amount of vector to be taken. According to our experiences, the optimum relation between size and amount of digested vector is as follows:

Size                                        Amount

<5 kb                                      50 ng

5-7.5 kb                                  75 ng

7.5-10 kb                                100 ng

> 10 kb                                   upto 150 ng

Taking amount of digested vector according to its size increases the ligation to a great extent and hence the cloning efficiency.

3)         Insert vs Vector Molar Ratio

Molar ratio plays a valuable role in the ligation of the fragments and hence in the cloning efficiency. But before moving further, let us see what molar ratio is? Molar ratio (sometimes also called as molar excess) is the amount of moles of insert per moles of vector molecule.

Generally, molar ratio is taken 3. That is, three insert fragments per vector molecule. It is a good practice to calculate the amount of insert in respect of vector and molar ratio prior to setting up the ligation reaction. The formula is:

ng of insert =  ^{amount of vector x molar ratio x size of insert} /size of the vector

For example, if the size of your vector is 6 kb and the size of insert is 1200 bp, then for setting up the ligation reaction you should take 75 ng of vector and 45 ng of insert.

However, if the difference between the size of insert and size of vector is more (e.g. insert=300 bp; vector=12000 bp), then molar ratio should be 5-10.

In order to accurately quantify the amount of eluted insert and vector molecules you should either use spectrophotometer or a nano-drop. However, we will not recommend you to estimate the amounts by running the samples in agarose gel. This does not allow accurate quantification of the fragment and hence results in lower ligation and cloning efficiency.

5)         Efficiency of the competent Cells

All well that ends well. This is also the case with gene cloning. If you have done all the things correct but you are not doing the last step properly, then you will ultimately ruin your whole hard work.

The ligation mix is used to transform E. coli competent cells and hence efficiency of the competent cells used significantly affects the cloning efficiency. We will recommend you to use high efficiency competent cells for your all cloning experiments. Always use the protocols which results in transformation efficiency of > 10⁷ cfu, for preparing competent cells.

However, if you are unable to prepare high efficiency competent cells, we will recommend you to go for commercially available competent cells. Nevertheless, you can easily prepare high efficiency competent cells, by going through our forthcoming article on competent cell preparation.

We hope that these informations will be helpful for your gene cloning experiments. However, if you have any further query regarding gene cloning, please feel free to post it in the comments.

cDNA Synthesis: Principle and Procedure

With the advancement in the field of genetic engineering, gene expression analysis has become an indispensable tool. Researchers are always keen to find out whether their gene of interest is expressing (turned on) or not (turned off). For this, the mRNA (messenger RNA) is located and quantified in the given sample. mRNAs carry the information coded by DNA and, thus, further gets translated to produce respective proteins.

RNAs are very unstable and fragile, and are very likely to degrade by the omnipresent RNases. In order to combat this, the biological informations encoded in mRNA are stored in more stable form of nucleic acid, i.e. DNA. Therefore, cDNA is prepared from RNA, which stores entire sequence of the mRNA. It is more convenient to work with cDNA as compared to mRNA. This cDNA can be further used for various subsequent molecular biology and genetic studies.

What is cDNA??

cDNA means complementary DNA or copy DNA. According to the central dogma of the molecular biology, DNA is transcribed into mRNA. Then mRNA gets translated to produce protein. Therefore, the flow of biological information is from DNA to RNA to protein.

However, sometimes the flow of information is from RNA to DNA (as in the case of some viruses, e.g. HIV). This conversion of RNA to DNA is aided by an enzyme known as Reverse Transcriptase (i.e. RNA-dependent DNA polymerase). The cDNA prepared can be single stranded or double stranded. Therefore, molecular biologists make use of reverse transcriptase to prepare cDNA from mRNA for the sake of convenience in the molecular studies.

Principle of cDNA synthesis

Mature (fully spliced) mRNA is used as a template for preparing cDNA. In fact, cDNA can be produced from any RNA molecule. This conversion is brought about by reverse transcriptase. cDNA can be obtained both from prokaryotes and eukaryotes.

Reverse transcriptase is a RNA-dependent DNA polymerase. It acts on a single strand of mRNA. Using mRNA as a template, reverse transcriptase produces its complementary DNA based on the pairing of RNA base pairs. This enzyme executes reactions in the same way as DNA polymerase. It also requires a primer with a free 3′-hydroxyl group.  For transcribing RNA having secondary structures, a reverse transcriptase with high temperature performance is recommended.

Procedure of cDNA synthesis

First of all, good quality intact mRNA or total RNA is isolated. Then, you need a few more reagents to prepare cDNA: dNTPs (dATP, dTTP, dCTP, dGTP), primers and reverse transcriptase.

In case of eukaryotic mRNAs, a poly-A tail is present at their 3′-ends. Therefore, a poly-T oligonucleotide is used as a primer. But certain modifications are needed when you use other RNAs which lack poly-A tail, e.g. prokaryotic mRNA, rRNA, RNA virus genomes, etc. In such cases, a poly-A tail is added to the 3′-end of the RNA. This makes it analogous to the eukaryotic mRNA.

The primer gets annealed to the 3′-end of the mRNA. Now, the 3′-end of the primer is extended with the help of the reverse transcriptase using mRNA strand as a template. This is known as “first strand reaction”. As a result of this, RNA-DNA hybrid molecule is produced. By the use of RNase H or alkaline hydrolysis, the RNA strand of this RNA-DNA hybrid molecule is digested. Now, the single stranded cDNA becomes free.

The reverse transcriptase used (most commonly used is Moloney Murine Leukemia Virus Reverse Transcriptase, MMLV RT) displays terminal transferase activity on reaching the end of the RNA template. It adds 3-5 residues (usually dC) to the 3′-terminal of the first strand cDNA. An oligo containing a stretch of G residues is used. This oligo gets annealed to the dC rich cDNA tail and serves as an extended template for reverse transcriptase. Now, the synthesis of the complementary strand of the first strand cDNA begins. This is called “second strand reaction”. Finally, a regular double stranded DNA is produced.

Types of primers used

Various types of primers can be used, in accordance to the requirements, to synthesize cDNA.

1) Oligo-dT primer- It is used when the mRNAs have poly-A tail, as in the case of eukaryotic mRNAs; or when a poly-A tail is attached to the existing RNA. Oligo-dT primer anneals to all the mRNAs simultaneously.

2) Sequence-specific primer- If you wish to generate cDNA from a particular population of mRNA among all the mRNAs, then sequence-specific primer is used. It will bind to a particular mRNA sequence only. This will give rise to a pure cDNA population generated from the desired mRNA. For designing sequence-specific primer, you must know the sequence of the mRNA of interest. Generally, the 3′-terminal sequence is preferred.

3) Random primer- A random primer cocktail is used to produce cDNA from all the mRNAs. The cDNAs produced are not full length. Random primer is extremely useful if production of the shorter cDNA fragments is desirable. Its use increases the probability of converting the entire 5′-end of the mRNA into the cDNA. In case of long mRNAs, reverse transcriptase is usually not able to reach the 5′-end. Therefore, random primer proves to be extremely advantageous in such cases.

Types of cDNA

cDNAs can be single stranded or double stranded. After the first strand reaction, cDNA obtained is single stranded. This single stranded cDNA can be converted to the double stranded form by second strand reaction. On the basis of the applications, single or double stranded form of the cDNA is used.

Applications of single stranded cDNA

1)      Single stranded cDNA is most commonly used for RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). RT-PCR is done for gene expression studies. It determines whether the gene of interest is expressed or not, and the level of its expression.

2)      It is also used to amplify particular gene of interest. For this, sequence-specific primers are used.

3)      Real-time PCR (also known as quantitative RT-PCR, qRT-PCR) also makes use of single stranded cDNA. It is done for performing gene expression analysis. As the amplification progresses, the amplicons can be visualized with the help of a fluorescent reporter molecular. It is highly sensitive and effective as compared to RT-PCR.

Applications of double stranded cDNA

1)      Double stranded cDNAs are used to clone them into the appropriate vector to prepare libraries of cDNA (i.e. cDNA libraries). These libraries contain all the mRNA sequences in the form of cDNA, which are all expressed in a cell.

2)      Double stranded form of a particular cDNA of interest can be cloned. Then, expression of the desired genes is allowed at the RNA and protein level for further study.

3)      Sequencing of the double stranded cDNA is carried out to obtain the expressed sequence tags (ESTs).

4)      They are also used for doing microarray for analysing global gene expression.

5)      Suppression subtractive hybridization (SSH) is also performed with double stranded cDNA. It is done to find out differential gene expression.

Lyophilization

Biological materials often are dried to stabilize them for storage or distribution. Lyophilization also called freeze-drying, is one method of drying biological materials that minimizes damage to its internal structure

Competitive ELISA

In competitive ELISA, unlabeled antibody is incubated in the presence of its antigen. Then these bound antibody/antigen complexes are then added to an antigen coated well. After washing, unbound antibodies are removed. The more analytes in the sample, the less antibodies will be able to bind to antigens in the well. The signal is then detected using labeled secondary antibodies and the decrease in signal is compared to a control. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.

Indirect ELISA

The indirect ELISA is used primarily to determine the strength and/or amount of antibody response in a sample. In the assay, the antigen of interested is immobilized by direct adsorption to the assay plate. Detection of the antigen can then be performed by using a matched set of primary antibody and conjugated secondary antibodies.

Sandwich ELISA

The Sandwich ELISA measures the amount of analyte between capture antibody and detection antibody. The analyte needs to have two different epitope sites available for antibody binding.

Ampicillin

Ampicillin is one of the most widely used antibiotic in molecular cloning, for the selection of transformants. It belongs to the penicillin group of antibiotics i.e. beta-lactam antibiotics. The only difference between penicillin and ampicillin is the presence of amino group in the latter.

Unlike penicillin which is effective against only gram positive bacteria, ampicillin is also effective against gram negative bacteria, e.g. E. coli. The amino group present in the side chain of the ampicillin renders it to penetrate the outer membrane of gram negative bacteria and hence enter into it. Therefore, it was one of the first broad spectrum penicillins to be used.

Mode of action

Like other beta-lactam antibiotics, ampicillin also inhibits cell wall synthesis in the bacteria. It basically inhibits the formation of glycan moiety of the peptidoglycan layer of the batcerial cell wall. The peptidoglycan is known to provide the rigidity to the bacterial cell wall.

Ampicillin is basically a competitive inhibitor of the peptidoglycan synthesizing enzyme transpeptidase. Inhibition of the enzyme, ultimately results in the lysis of the bacterial cells.

Ampicillin resistance gene

The gene responsible for conferring ampicillin resistance is called bla gene and codes for a TEM1 ?-lactamase enzyme.  The TEM1 ?-lactamase hydrolyzes the ?-lactam ring of the ampicillin. This results in the inactivation of the antibiotic. The enzyme is usually secreted into the periplasmic space where it catalyzes the hydrolysis reaction.

TEM1 ?-lactamase is the major ?-lactamase responsible for ampicillin resistance in various gram negative bacteria including E. coli. As a result, this gene is widely used to confer resistance against ampicillin. There are a variety of vectors using bla gene to confer resistance against ampicillin for the selection of transformed E. coli.

Preparing the ampicillin stock solution

The stock solution of ampicillin is prepared in water. Generally, ampicillin sodium salt is used for its better solubility. The concentration of stock solution is generally in the range of 50-100mg/ml. We usually prepare 100mg/ml stock solutions.

Weigh 100mg of ampicillin sodium salt, dissolve it in 1ml MQ grade water and filter sterilize it by using 0.22µm filter. Store the stock at -20?C.

You should note that pH of the water has great impact on the stability of the antibiotic. It is better to use pH 6.8-7.0, of water for preparing the stock. Moreover the stock can be stored for one month only. After that the antibiotic starts to degrade.

Working concentration of ampicillin in media

For broth or agar plates the concentration of ampicillin is kept between 50-100µg/ml.

Problem of satellite colonies

As the beta lactamase is secreted in the periplasm, it degrades the ampicillin nearby the transformed cells. This results in the growth of small untransformed colonies in close proximity of the transformed one. These colonies are called as satellite colonies.

One way to deal with satellite colonies is to incubate the plates for less than 14 hours. Another way is to increase the concentration of ampicillin in the plates. We generally use 100µg/ml of ampicillin in the plates. But here also it is better to incubate the plates for less than 16 hours.

9 Factors affecting DNA extraction from agarose gel

Agarose gel electrophoresis is a method of choice for the identification, purification, and separation of the DNA fragments. DNA fragments from the gel are routinely extracted for various downstream processing. These include, cloning, radio-labeling, in vitro transcription, microinjection and sequencing of the DNA molecules.

Gel extraction of DNA fragments is mainly done to remove proteins and salts that incorporate from certain reactions. Therefore, in order to use the DNA fragments for downstream processing, these components musts be removed. For example, a PCR amplification or restriction enzyme digestion reaction contains factors which inhibit further applications of the DNA fragment.

There are various methods employed for the extraction of DNA fragments from agarose gel. Among the methods used, silica-membrane containing spin-column based DNA extraction is the most widely used. This is the quickest method to effectively purify DNA fragments from agarose gels.

However, DNA extraction using this method is not always efficient and depends on following factors:

1) Size of the DNA fragment

The first factor affecting the extraction of DNA fragment from the agaorose gel is the size of the DNA fragment itself. It has been observed that DNA fragments of size 500-5000bp are extracted most efficiently from the agarose gels. However, DNA fragments smaller than 500bp or lager than 5000bp, are poorly recovered from the gel.

2) Concentration of agarose in the gel

Concentration of agarose in the gel has a significant effect on the extraction of DNA fragments. It has been observed that recovery of DNA fragments is inversely proportional to the concentration of agarose in the gel. As the concentration of agarose in the gel increases, recovery of DNA molecules decreases. Therefore, in order to carryout good recovery of DNA fragments, less concentration of agarose is used in the gel.

Generally, for gel extraction, 0.7-1% agarose gels are prepared. However, if the size of the DNA fragment is less than 300bp you will have to use higher concentration of agarose (1.2-1.5%) in the gel. We routinely use 0.7% agarose gels for extracting DNA molecules.

3) Voltage applied to the gel

The applied voltage also significantly affects the extraction of DNA fragments. In order to extract any DNA molecule from the gel you need to excise it out. For this you need a sharp and distinct band of specific DNA in the gel. Therefore, to get a sharp band, the gel has to be run using the appropriate voltage.

It has been observed that 3-5V/cm is the optimum voltage for DNA extraction. Increasing or decreasing the voltage will result in poor resolution and hence the poor recovery of the DNA fragments. For extracting the DNA fragments, we generally apply a voltage of 3V/cm to the gel.

4) Cutting of the gel slice

Cutting the gel slice also has a significant effect on the recovery of the DNA molecules. You should cut the gel slice just adjacent to the band. Moreover, extra gel portions if any should be carefully removed. It has been found that extra gel portions greatly reduce the recovery of DNA molecules.

In addition, you should try to cut small gel slices for extraction purpose. Lager gel slices incompletely solubilize and hence results in the poor recovery of the DNA molecules.

5) pH of the extraction buffer

As the adsorption of DNA fragments to silica membranes largely depends on pH, therefore, extraction of DNA fragments is significantly affected by the pH of the extraction buffer.  It has been observed that maximum adsorption occurs at pH less than 7.5. As the pH of the buffer increases, adsorption of DNA to silica membrane is reduced drastically.

In order to monitor the pH of the extraction buffer, many commercially available kits add indicators to the extraction buffer. If the pH increases, color of the buffer changes, thereby enabling easy monitoring of the pH.

There are certain reasons for the increase of pH of the extraction buffer. For example, agarose gel is having incorrectly prepared high pH buffer, or the electrophoresis buffer had been repeatedly used, etc. In these cases, the pH of the extraction buffer can easily be corrected by adding a small volume of 3 M sodium acetate, pH 5.2, before proceeding.

6) Residual ethanol

After the adsorption of the DNA molecules to the silica membrane, the membrane is washed with solutions having 70% ethanol. However, before the addition of elution buffer to the membrane, the residual ethanol must be removed. It has been observed that presence of residual ethanol on the silica membrane greatly reduces the recovery of the DNA fragments.

In order to remove the residual ethanol the best way is to centrifuge the column for two minutes at maximum rpm. However, we have observed that doing so doesn’t completely remove the residual ethanol. Therefore, you should keep the column at room temperature for a while to remove the residual ethanol. But be careful, as over-drying the membrane will, in turn, result in lower recovery of the DNA fragments.

7) pH and salt concentration of the elution buffer

Elution efficiency of the DNA fragments from silica membranes strongly depends on the salt concentration and pH of the elution buffer. It has been found that most efficient elution is carried out under alkaline pH (7.5-8.5) and low salt concentrations.

Generally, the DNA fragments are eluted using 10 mM Tris-Cl, pH 8.5. However, TE buffer is not recommended for the elution of the DNA because some downstream processing may get inhibited by the presence of EDTA. Apart from 10 mM Tris-Cl, pH 8.5, MQ grade water can also be used to elute the DNA fragments. However, you will have to make sure that the pH of the water remains in the range of 7.5-8.5. Moreover, recovery of DNA fragments will be less if water is used instead of the elution buffer.

8) Volume of elution buffer

Generally, elution of DNA fragments in a spin column based extraction is carried out in small volumes. However, the volume of the elution buffer plays a significant role in the recovery of the DNA molecules. It has been observed that most efficient elution is carried out using 20-50µl of the elution buffer.

Using elution buffer less than 20µl will result in poor recovery of the DNA fragments. However, using lager volumes will result in the elution of less concentrated DNA fragments. For various purposes, we generally elute the DNA fragments in 25µl of elution buffer.

9) Incubation time of the buffer on the column

Since the main purpose of the elution buffer is to provide a medium in which the DNA fragments will be dissolved. Therefore, incubating the spin column after the addition of the elution buffer has a significant role in the recovery of the DNA molecules. Generally, it is a good practice to incubate the column for 2-5 minutes prior to centrifugation.

You can clearly see that there are a number of factors influencing the extraction of DNA fragments from agarose gel using spin column based method. Each of the above listed factors should be properly considered in order to achieve efficient extraction of the DNA.

Electricity Producing Bacteria

Genetic Engineering in Livestock

Recently, the first "zoofarming" product has reached market approval: it is a recombinant human protein for medical use that is produced in the milk of transgenic goats. In addition, other transgenic animals, including faster-growing salmon and „environmentally friendly" pigs with reduced levels of phosphate in their feces are awaiting regulatory approval. These are only some examples of upcoming applications of genetic engineering in farm animals. Other potential applications include traditional breeding goals such as higher milk or meat yields, leaner meat, and disease resistance. While genetic engineering in livestock opens a huge range of possibilities, it also brings about concerns of safety and justification: does genetic engineering affect animal welfare? Is it safe and morally acceptable to apply genetic engineering to farm animals for the various purposes that are envisaged?
It is against this background that the Europäische Akademie GmbH and the Berlin-Brandenburgische Akademie der Wissenschaften addressed the topic of transgenic farm animals in an interdisciplinary symposium in 2007. In these proceedings the following topics are covered: an analysis of the state of the art of the technology and its applications, an introduction to the specific application zoopharming (including its historical industrial development and the market for biopharmaceuticals), an assessment of ethical aspects, and considerations regarding the investigation of animal welfare implications of livestock biotechnology. The proceedings address science, industry, politics and the general public interested in the chances and risks of this upcoming field of biotechnology.

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Angiogenesis

Angiogenesis is a normal process in growth and development, as well as in wound healing. However, this is also a fundamental step in the transition of tumors from a dormant state to a malignant state.

Sprouting angiogenesis was the first identified form of angiogenesis. It occurs in several well-characterized stages. First, biological signals known as angiogenic growth factors activate receptors present on endothelial cells present in pre-existing venular blood vessels. Second, the activated endothelial cells begin to release enzymes called proteases that degrade the basement membrane in order to allow endothelial cells to escape from the original (parent) vessel walls. The endothelial cells then proliferate into the surrounding matrix and form solid sprouts connecting neighboring vessels. As sprouts extend toward the source of the angiogenic stimulus, endothelial cells migrate in tandem, using adhesion molecules, the equivalent of cellular grappling hooks, called integrins. These sprouts then form loops to become a full-fledged vessel lumen as cells migrate to the site of angiogenesis. Sprouting occurs at a rate of several millimeters per day, and enables new vessels to grow across gaps in the vasculature. It is markedly different from splitting angiogenesis, however, because it forms entirely new vessels as opposed to splitting existing vessels.

Types of angiogenesis

Sprouting angiogenesis
Intussusceptive angiogenesis

Intussusception, also known as splitting angiogenesis, was first observed in neonatal rats. In this type of vessel formation, the capillary wall extends into the lumen to split a single vessel in two. There are four phases of intussusceptive angiogenesis. First, the two opposing capillary walls establish a zone of contact. Second, the endothelial cell junctions are reorganized and the vessel bilayer is perforated to allow growth factors and cells to penetrate into the lumen. Third, a core is formed between the two new vessels at the zone of contact that is filled with pericytes and myofibroblasts. These cells begin laying collagen fibers into the core to provide an extracellular matrix for growth of the vessel lumen. Finally, the core is fleshed out with no alterations to the basic structure. Intussusception is important because it is a reorganization of existing cells. It allows a vast increase in the number of capillaries without a corresponding increase in the number of endothelial cells. This is especially important in embryonic development as there are not enough resources to create a rich microvasculature with new cells every time a new vessel develops.

Modern Terminology of Angiogenesis

Besides the differentiation between “Sprouting angiogenesis” and “Intussusceptive angiogenesis” there exists the today more common differentiation between the following types of angiogenesis:

Vasculogenesis – Formation of vascular structures from circulating or tissue-resident endothelial stem cells (angioblasts), which proliferate into de-novo endothelial cells. This form particularly relates to the embryonal development of the vascular system.

Angiogenesis – Formation of thin-walled endothelium-lined structures with/without muscular smooth muscle wall and pericytes (fibrocytes). This form plays an important role during the adult life span, also as "repair mechanism" of damaged tissues.

Arteriogenesis – Formation of medium-sized blood vessels possessing tunica media plus adventitia.

Because it turned out that even this differentiation is not a sharp one, today quite often the term “Angiogenesis” is used summarizing all different types and modifications of arterial vessel growth.

Therapeutic angiogenesis

Therapeutic angiogenesis is the application of specific compounds which may inhibit or induce the creation of new blood vessels in the body in order to combat disease. The presence of blood vessels where there should be none may affect the mechanical properties of a tissue, increasing the likelihood of failure. The absence of blood vessels in a repairing or otherwise metabolically active tissue may retard repair or some other function. Several diseases (eg. ischemic chronic wounds) are the result of failure or insufficient blood vessel formation and may be treated by a local expansion of blood vessels, thus bringing new nutrients to the site, facilitating repair. Other diseases, such as age-related macular degeneration, may be created by a local expansion of blood vessels, interfering with normal physiological processes.

The modern clinical application of the principle “angiogenesis” can be divided into two main areas: 1. Anti-angiogenic therapies (historically, research started with); 2. Pro-angiogenic therapies. Whereas anti-angiogenic therapies are trying to fight cancer and malignancies(because tumors, in general, are nutrition- and oxygen-dependent, thus being in need of adequate blood supply), the pro-angiogenic therapies are becoming more and more important in the search of new treatment options for cardiovascular diseases (the number one cause of death in the Western world). One of the world-wide first applications of usage of pro-angiogenic methods in humans was a German trial using fibroblast growth factor 1 (FGF-1) for the treatment of coronary artery disease. Today, clinical research is ongoing in various clinical trials to promote therapeutic angiogenesis for a variety of atherosclerotic diseases, like coronary heart disease, peripheral arterial disease, wound healing disorders, etc.

Also, regarding the “mode of action”, pro-angiogenic methods can be differentiated into three main categories: 1. Gene-therapy; 2. Protein-therapy (using angiogenic growth factors like FGF-1 or vascular endothelial growth factor, VEGF); 3. Cell-based therapies.

There are still serious, unsolved problems related to gene therapy including: 1. Difficulty integrating the therapeutic DNA (gene) into the genome of target cells; 2. Risk of an undesired immune response; 3 Potential toxicity, immunogenicity, inflammatory responses and oncogenesis related to the viral vectors; and 4. The most commonly occurring disorders in humans such as heart disease, high blood pressure, diabetes, Alzheimer’s disease are most likely caused by the combined effects of variations in many genes, and thus injecting a single gene will not be beneficial in these diseases. In contrast, pro-angiogenic protein therapy uses well defined, precisely structured proteins, with previously defined optimal doses of the individual protein for disease states, and with well-known biological effects. On the other hand, an obstacle of protein therapy is the mode of delivery: oral, intravenous, intra-arterial, or intramuscular routes of the protein’s administration are not always as effective as desired; the therapeutic protein can be metabolized or cleared before it can enter the target tissue. Cell-based pro-angiogenic therapies are still in an early stage of research – with many open questions regarding best cell types and dosages to use.

FGF

The fibroblast growth factor (FGF) family with its prototype members FGF-1 (acidic FGF) and FGF-2 (basic FGF) consists to date of at least 22 known members. Most are 16-18 kDa single chain peptides and display high affinity to heparin and heparan sulfate. In general, FGFs stimulate a variety of cellular functions by binding to cell surface FGF-receptors in the presence of heparin proteoglycans. The FGF-receptor family is comprised of seven members and all the receptor proteins are single chain receptor tyrosine kinases that become activated through autophosphorylation induced by a mechanism of FGF mediated receptor dimerization. Receptor activation gives rise to a signal transduction cascade that leads to gene activation and diverse biological responses, including cell differentiation, proliferation, and matrix dissolution – thus initiating a process of mitogenic activity critical for the growth of endothelial cells, fibroblasts, and smooth muscle cells. FGF-1, unique among all 22 members of the FGF family, can bind to all seven FGF-receptor subtypes, making it the broadest acting member of the FGF family, and a potent mitogen for the diverse cell types needed to mount an angiogenic response in damaged (hypoxic) tissues, where up regulation of FGF-receptors occurs. FGF-1 stimulates the proliferation and differentiation of all cell types necessary for building an arterial vessel, including endothelial cells and smooth muscle cells; this fact distinguishes FGF-1 from other pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF) which primarily drives the formation of new capillaries.

Until now (2007), three human clinical trials have been successfully completed with FGF-1 in which the angiogenic protein was injected directly into the damaged heart muscle. Also, one additional human FGF-1 trial has been completed to promote wound healing in diabetics with chronic wounds.

Besides FGF-1, one of the most important functions of also fibroblast growth factor-2 (FGF-2 or bFGF) is the promotion of endothelial cell proliferation and the physical organization of endothelial cells into tube-like structures, thus promoting angiogenesis. FGF-2 is a more potent angiogenic factor than VEGF or PDGF (platelet-derived growth factor), however, less potent than FGF-1. As well as stimulating blood vessel growth, aFGF (FGF-1) and bFGF (FGF-2) are important players in wound healing. They stimulate the proliferation of fibroblasts and endothelial cells that give rise to angiogenesis and developing granulation tissue, both increase blood supply and fill up a wound space/cavity early in the wound healing process.

VEGF

VEGF (Vascular Endothelial Growth Factor) has been demonstrated to be a major contributor to angiogenesis, increasing the number of capillaries in a given network. Initial in vitro studies demonstrated that bovine capillary endothelial cells will proliferate and show signs of tube structures upon stimulation by VEGF and bFGF, although the results were more pronounced with VEGF. Upregulation of VEGF is a major component of the physiological response to exercise and its role in angiogenesis is suspected to be a possible treatment in vascular injuries. In vitro studies clearly demonstrate that VEGF is a potent stimulator of angiogenesis because in the presence of this growth factor plated endothelial cells will proliferate and migrate, eventually forming tube structures resembling capillaries. VEGF causes a massive signaling cascade in endothelial cells. Binding to VEGF receptor-2 (VEGFR-2) starts a tyrosine kinase signaling cascade that stimulates the production of factors that variously stimulate vessel permeability (eNOS, producting NO), proliferation/survival (bFGF), migration (ICAMs/VCAMs/MMPs) and finally differentiation into mature blood vessels. Mechanically, VEGF is upregulated with muscle contractions as a result of increased blood flow to affected areas. The increased flow also causes a large increase in the mRNA production of VEGF receptors 1 and 2. The increase in receptor production means that muscle contractions could cause upregulation of the signaling cascade relating to angiogenesis. As part of the angiogenic signaling cascade, NO is widely considered to be a major contributor to the angiogenic response because inhibition of NO significantly reduces the effects of angiogenic growth factors. However, inhibition of NO during exercise does not inhibit angiogenesis indicating that there are other factors involved in the angiogenic response.

Angiopoietins

The angiopoietins, Ang1 and Ang2, are required for the formation of mature blood vessels, as demonstrated by mouse knock out studies . Ang1 and Ang2 are protein growth factors which act by binding their receptors, Tie-1 and Tie-2; while this is somewhat controversial, it seems that cell signals are transmitted mostly by Tie-2; though some papers show physiologic signaling via Tie-1 as well. These receptors are tyrosine kinases. Thus, they can initiate cell signaling when ligand binding causes a dimerization that initiates phosphorylation on key tyrosines.

MMP

Another major contributor to angiogenesis is matrix metalloproteinase (MMP). MMPs help degrade the proteins that keep the vessel walls solid. This proteolysis allows the endothelial cells to escape into the interstitial matrix as seen in sprouting angiogenesis. Inhibition of MMPs prevents the formation of new capillaries. These enzymes are highly regulated during the vessel formation process because destruction of the extracellular matrix would decrease the integrity of the microvasculature.

Applications of Angiogenesis
Tumor angiogenesis

Cancer cells are cells that have lost their ability to divide in a controlled fashion. A tumor consists of a population of rapidly dividing and growing cancer cells. Mutations rapidly accrue within the population. These mutations (variation) allow the cancer cells (or sub-populations of cancer cells within a tumor) to develop drug resistance and escape therapy. Tumors cannot grow beyond a certain size, generally 1-2 mm³, due to a lack of oxygen and other essential nutrients.

Tumors induce blood vessel growth (angiogenesis) by secreting various growth factors (e.g. Vascular Endothelial Growth Factor or VEGF). Growth factors, such as bFGF and VEGF can induce capillary growth into the tumor, which some researchers suspect supply required nutrients -- allowing for tumor expansion. On 18 July 2007 it was discovered that cancerous cells stop producing the anti-VEGF enzyme PKG. In normal cells (but not in cancerous ones), PKG apparently limits beta-catenin which solicits angiogenesis.Other clinicians believe that angiogenesis really serves as a waste pathway, taking away the biological end products put out by rapidly dividing cancer cells. In either case, angiogenesis is a necessary and required step for transition from a small harmless cluster of cells, often said to be about the size of the metal ball at the end of a ball-point pen, to a large tumor. Angiogenesis is also required for the spread of a tumor, or metastasis. Single cancer cells can break away from an established solid tumor, enter the blood vessel, and be carried to a distant site, where they can implant and begin the growth of a secondary tumor. Evidence now suggests that the blood vessel in a given solid tumor may in fact be mosaic vessels, comprised of endothelial cells and tumor cells. This mosaicity allows for substantial shedding of tumor cells into the vasculature. The subsequent growth of such metastases will also require a supply of nutrients and oxygen or a waste disposal pathway.

Endothelial cells have long been considered genetically more stable than cancer cells. This genomic stability confers an advantage to targeting endothelial cells using antiangiogenic therapy, compared to chemotherapy directed at cancer cells, which rapidly mutate and acquire 'drug resistance' to treatment. For this reason, endothelial cells are thought to be an ideal target for therapies directed against them. Recent studies by Klagsbrun, et al. have shown, however, that endothelial cells growing within tumors do carry genetic abnormalities. Thus, tumor vessels have the theoretical potential for developing acquired resistance to drugs. This is a new area of angiogenesis research being actively pursued.

Angiogenesis research is a cutting edge field in cancer research, and recent evidence also suggests that traditional therapies, such as radiation therapy, may actually work in part by targeting the genomically stable endothelial cell compartment, rather than the genomically unstable tumor cell compartment. New blood vessel formation is a relatively fragile process, subject to disruptive interference at several levels. In short, the therapy is the selection agent which is being used to kill a cell compartment. Tumor cells evolve resistance rapidly due to rapid generation time (days) and genomic instability (variation), whereas endothelial cells are a good target because of a long generation time (months) and genomic stability (low variation).

This is an example of selection in action at the cellular level, using a selection pressure to target and differentiate between varying populations of cells. The end result is the extinction of one species or population of cells (endothelial cells), followed by the collapse of the ecosystem (the tumor) due either to nutrient deprivation or self-pollution from the destruction of necessary waste pathways.

Angiogenesis-based tumour therapy relies on natural and synthetic angiogenesis inhibitors like angiostatin, endostatin and tumstatin. These are proteins that mainly originate as specific fragments pre-existing structural proteins like collagen or plasminogen.

Recently, the 1st FDA-approved therapy targeted at angiogenesis in cancer came on the market in the US. This is a monoclonal antibody directed against an isoform of VEGF. The commercial name of this antibody is Avastin, and the therapy has been approved for use in colorectal cancer in combination with established chemotherapy.

Angiogenesis for cardiovascular disease

Angiogenesis represents an excellent therapeutic target for the treatment of cardiovascular disease. It is a potent, physiological process that underlies the natural manner in which our bodies respond to a diminution of blood supply to vital organs, namely the production of new collateral vessels to overcome the ischemic insult. Perhaps the greatest reason for these trials’ failure to achieve success is the high occurrence of the “placebo effect” in studies employing treadmill exercise test readout. Thus, even though a majority of the treated patients in these trials experience relief of such clinical symptoms such as chest pain (angina), and generally performed better on most efficacy readouts, there were enough “responders” in the blinded placebo groups to render the trial inconclusive. In addition to the placebo effect, more recent animal studies have also highlighted various factors that may inhibit an angiogenesis response including certain drugs, smoking, and hypercholesterolemia.

Although shown to be relatively safe therapies, not one angiogenic therapeutic has yet made it through the gauntlet of clinical testing required for drug approval. By capitalizing on the large database of what did and did not work in previous clinical trials, results from more recent studies with redesigned clinical protocols give renewed hope that angiogenesis therapy will be a treatment choice for sufferers of cardiovascular disease resulting from occluded and/or stenotic vessels.

Early clinical studies with protein-based therapeutics largely focused on the intravenous or intracoronary administration of a particular growth factor to stimulate angiogenesis in the affected tissue or organ. Most of these trials did not achieve statistically significant improvements in their clinical endpoints. This ultimately led to an abandonment of this approach and a widespread belief in the field that protein therapy, especially with a single agent, was not a viable option to treat ischemic cardiovascular disease. However, the failure of gene- or cell-based therapy to deliver, as of yet, a suitable treatment choice for diseases resulting from poor blood flow, has led to a resurgence of interest in returning to protein-based therapy to stimulate angiogenesis. Lessons learned from earlier protein-based studies, which indicated that intravenous or intracoronary delivery of the protein was not efficacious, have led to completed and ongoing clinical trials in which the angiogenic protein is injected directly into the beating ischemic heart.

Such localized administration of the potent angiogenic growth factor, human FGF-1, has recently given promising results in clinical trials in no-option heart patients. Angiogenesis was documented by angiographically visible “blushing”, and functional exercise tests were also performed on a subset of patients. The attractiveness of protein therapy is that large amounts of the therapeutic agent can be injected into the ischemic area of interest, to pharmacologically start the process of blood vessel growth and collateral arteries’ formation. In addition, from pharmacokinetic data collected from the recent FGF-1 studies in the human heart, it appears that FGF-1, once it exits the heart is cleared in less than three hours from the circulation. This would presumably prevent FGF-1 from stimulating unwanted angiogenesis in other tissues of the bodies where it could potentially cause harm, such as the retina and in the kidneys. No serious adverse events have yet to be noted in any of the completed or ongoing clinical trials in which the FGF-1 protein is utilized as the therapeutic agent tom stimulate angiogenesis