Monday, October 28, 2013

Isolation of monocytes from PBMC (Peripheral Blood Mononuclear Cells) - Principle and protocol

Whenever I'm made to realise that I'm not clear enough or good at something, I try to make myself clear with it. It happened today, during my laboratory examination, I was asked to perform monocyte isolation from a given blood sample, but, unfortunately, I was not very clear with the principle behind it.(but, still I managed to complete the experiment as I know the protocol, but, knowing the principle behind each step of the protocol clearly is very important, isn't it?). But, nothing is wrong in it, I made myself clear with it now. That's good, right?

So, let me share with you some basic principle and protocol for isolating monocyte from blood sample.

For isolating monocytes, initially we must isolate PBMC (Peripheral bood mono nuclear cells) from the blood sample. Here, let us make few terminologies clear before starting with the principle.

Peripheral blood sample - It is the blood sample obtained from acral areas of body (in general, it is the blood collected from hands by your doctor for doing blood test).

Peripheral blood mononuclear cells (PBMCs) - It is the cells which have a prominent round nucleus which is present in the peripheral blood sample. Note, all the blood cells have a single nucleus, i.e, mono nucleus, but, neutrophil, basophil and eosinophils have lobed nucleus, i.e. they are polymorphic. Lymphocytes and monocytes have round nucleus and they are referred as PBMCs.

Ficoll hypaque - It is a polysaccharide which is soluble in aqueous solutions and widely used for performing separations based on density. Here, we can isolate PBMC's from RBC's and other blood cells based on the difference in their density.

Percoll Gradient - Percoll is silica particles coated with PVP (polyvinyl pyrollidine) and preferred for biological isolations because of its characteristics like non toxicity and low osmolarity. Besides it's use in monocyte isolation it is also used in sperm selection in assisted reproduction where healthy and active sperm cells can be selected using Percoll gradient.

Isolation of PBMCs

Protocol in short - (if 1ml of blood sample is taken, dilute with 1ml of 1X PBS before starting, when you use 10XPBS it is reported that the separation is not efficient, problem occurs while overlaying ficoll hypaque)

Take 2ml of ficoll hypaque for isolating PBMCs from 4ml of diluted blood (2ml blood diluted with 2ml of 1X PBS) in a falcon (always maintain 1:2 ratio of Ficoll and blood sample). Overlay the Ficoll hypaque with diluted 4ml of blood sample using pipette (do it slowly to avoid mixing of the blood with ficoll due to force given during addition). Centrifuge this at 1400rpm for 30 minutes at 20 degree C (brake off) 

After centrifugation, you can find buffy coat containing PBMCs over the Ficoll layer, as shown in the figure below: (hisep is commercial name of Ficoll)


Carefully the plasma layer must be discarded and the buffy coat is collected in a separate clean falcon (must be done with care to avoid mixing of layers). The separation of the cells is based on their density, Platelets are the least dense cells and they top the layers, followed by PBMCs which are also not dense enough to cross the Ficoll whereas RBCs and other WBCs are dense enough to cross the Ficoll. 

The separated buffy coat is further washed with suitable medium at 1400rpm, 15minutes, 20 degree C (brake on). Again resuspend the pellet in appropriate medium and do Trypan Blue viability check assay and count the viable cells and  find the number of viable cells.

And, normally the yield will be 0.5to 3 *10^6 cells/ ml.

Isolation of monocytes using Percoll gradient

Prepare Percoll gradient (using Standard Isotonic Percoll 9parts Percoll + 1 part 10X PBS) by using 0.15M NaCl. Generally, 60% (lower layer), 45% (middle layer) and 35% (top layer) percoll solutions are prepared for preparing the gradient. For a concentration of 3*10^7 cells lower and top layer of 2.5ml and middle layer of 5ml can be used. This is prepared by layering successive layers in a falcon, if required, centrifuged. 

Then overlay the gradient with PBMC and after centrifugation at 1400 rpm for 30 minutes, monocytes can be obtained from the middle layer.

Hope, this helped you. any mistakes? Doubts? kindly comment! 

Thursday, October 24, 2013

Lowry Assay Principle and procedure

Though there are several protein assays available, the most preferred one in many laboratories is "Lowry assay". It is effective in the concentration range of 0.01 mg/ml to 1 mg/ml. And, as an additional info, the paper published describing the procedure and principle of Lowry Assay is the most cited paper in the scientific history. (Feeling like, "Wow! I want to publish one to compete with Oliver.H. Lowry"???  :P)

Why Lowry? 

Though there are several other protein assays, mostly Lowry assay is used in many laboratories. The reasons for preferring Lowry are: sensitivity of the assay, highly reproducible, cost effective, easy to perform. Biuret assay is generally used for higher protein concentrations like tissue samples but, Lowry for less concentrated samples and hence used in most of the molecular biology laboratories where there will be need for assaying comparatively less concentrated protein samples (in most cases where we attempt to produce enzymes).
Other assays like Bicinchoninic Acid and Dye binding assays are used in only specific circumstances and mostly not preferred due to its sensitiveness to contaminants like carbohydrates, lipids, etc.,

 Principle behind Lowry's Assay for protein with procedure

The reactions that occur in Lowry assay are binding of Copper to the Nitrogen in the peptide. And, the phosphomolybdic tungstic acid in the Folin Ciocalteau reagent gets reduced to hetero poly-molybdenum blue by the copper catalyzed oxidation of aromatic amino acids in the peptide, in alkaline conditions. The assay must be done at the pH of 10 to 10.5 as it is sensitive to pH changes.


Reagents Required:

A) 2% of sodium Carbonate (50 ml) + 0.1 N NaOH solution (50 ml)
B) 10 ml of 1.56 CuSo4 + 10 ml of 2.37% Sodium potassium tartarate

Lowry's Reagent = 2 ml of (B) + 100 ml of (A)
Folin's Reagent what we use in our laboratory is ready made one (2 N) which is just diluted and made as 1 N for use. (by mixing with equal volume of water)

For constructing Standard curve, BSA is generally used. Stock of 1 mg/ml is required. 

Note: Prepare all the reagents in distilled water. And, prepare the reagents freshly before use, just before use, to avoid precipitation of the salt added, also mix the reagents A and B only before use. 

Procedure:
  • First the BSA stock is diluted for standard curve construction. Let the total volume be 1 ml, so for preparing a concentration of 0.05 ml, take 0.05 ml of 1 mg/ml BSA stock and mix with 0.95 ml of distilled water.
  • Similarly, we can prepare various standard concentrations like 0.1,0.2,0.4,0.6,0.8,1 mg/ml for  total volume of 1 ml by mixing 0.1, 0.2,0.4,0.6,0.8,1 ml of stock BSA with 0.9,0.8,0.6,0.4,0.2,0 ml of distilled water respectively. 
  • From this prepared standard concentrations of BSA, 0.2 ml must be taken for assay. For example, from the 1 ml of 0.05 mg/ml BSA prepared 0.2 ml must be taken in a separate test tube. Similarly, 0.2 ml must be taken from all the other standards.
  • Then, 2 ml of Lowry's reagent must be added to each of this 0.2 ml sample and incubated for 10 minutes.
  • Then, 0.2 ml of Folin's reagent is added to each of the incubated tubes and incubated for 30 minutes.
  • After 30 minutes, the tubes will have blue colored solution, which is further read at  660 nm in Spectrophotometer. 
  • Graph should be plotted with absorbance in y axis and concentration in X axis. 
And, using this standard graph, we can determine the concentration of unknown sample by extrapolation.

This is a very easy assay which I learnt in the very beginning  of my course, but, still a very useful one and I'll be using it even after years I believe. 

You know, I feel the major disadvantage of this Lowry assay is that you need to spend at least two hours to complete it including reagent preparation and incubation. (Sometimes, I feel like, oh, no, incubation for 30 minutes!How good it would be if there is no need for incubation?) And, when you don't have that much time, what will you do for assaying your protein? 

It's simple, just measure absorbance at 260 nm and also 280 nm. Using the formula,

Protein (mg/ml) = 1.55 *(Absorbance at 280 nm) - 0.76 * (Absorbance at 260 nm).

This can be used when you have a spec which operates in UV range. No need for doing Lowry Assay, Yippee :P 

Hope, this helped you :) Found any mistakes? Doubts? Let me know with your comments!

Wednesday, October 2, 2013

Cre lox system - Basics

It's been a long time, since I wrote a post here. Finally, final year of my course and so very busy :) with books! And now, got time to share with you.

As you know already (don't know?, then get to know :P ), I use to have favorite subjects in each semester, third semester it was microbiology, then in fourth semester, it was cell biology, fifth was molecular biology, sixth was genetic engineering and now i'm in seventh semester, but, you know what, I don't have a favorite subject!!! Don't worry that I lost interest, instead, I got 3 favorite subjects - Immunology, Animal biotechnology and Plant Biotechnology!

It's awesome this time to have more than one favorite subject and here I'm gonna tell you something about, cre-lox system which is most widely used for making modifications (mostly deletions). These modifications can be done at a specific tissue alone by using a tissue specific promoter i.e., you can selectively knock out a particular gene in particular cells (for eg: hepatocytes) alone.

Cre recombinase is a protein which could bind with specific sequences called loxP sequences. To explain it in a simple way, the cre recombinase binds at loxP sites on either side of your gene (which is to be deleted) and makes a cut! This leads to removal of the gene and further the break is repaired using the DNA ligase of host system.

A single loxP site will have 13bp on either side of middle 8bp. The cre recombinase protein binds with this 13bp sequences forming dimer i.e., one loxP site contributes for a dimer (containing 2 cre recombinase units). Similarly, loxP on the other side of the target gene will contribute for a dimer formation. Then these two dimers will form tetramer (by folding the DNA strand as shown in figure. Then, cutting at the tetramer site happens leading to either translocation, inversion or deletion depending on the direction of the loxP site (i.e whether it is direct or invert repeat on either side of the target gene or located on different chromosomes)

                         13bp                          8bp                  13bp
             ATAACTTCGTATA -NNNTANNN-TATACGAAGTTAT





This insertion, deletion and translocation could be done specifically at a particulat type of cell by using specific promoters. For example: To delete a particular gene in kidney cells alone, one could use kidney specific promoter for controlling the production of cre recombinase, so that cre recombinase will be expressed only in kidney cells. The molecular mechanisms of this deletions and recombinations invloves holliday junction formation. But, to be frank, i'm not clear or good at this holliday junction.

I''m trying to understand the molecular mechanism behind this, and, once I'm clear with it, I'll share it with you.

Understood the cre-loxp system? Got  any doubt? anything wrong in my explanation? Kindly comment and I would try to reply or correct as soon as possible.


Tuesday, August 13, 2013

Diagnose your disease!

Let it be any disease, ranging from a fever (which is the symptom of many diseases) to HIV, diagnosis at an early stage is very important for giving specific treatment.Whenever I go to a doctor, he asks me things like, stomach pain, fever, body pain, head ache, running nose, throat pain etc., then, he checks pulse, then he uses his stethoscope, he then gives all the possible antibiotics! Yes, really, this happens in most cases. Then, if my problem is not fixing up in a week, he'll ask me to take a blood test, this, that, and, all!

Nothing wrong in this, because he can't ask everyone coming to him to take blood test at the very first sitting, then, no one will visit him back saying he sucks out blood all the time! But, it is okay to have a blood test after a week, but, not after a month! Diagnosing a disease after it had reached a severe stage is comparatively less significant than diagnosing at an early stage. So, how to diagnose?

Antigen - Antibody!

Antigen and Antibody interactions are mainly used for diagnosing most of the diseases. When you give your blood sample to your doctor, he gives it to his lab, there they check for the presence of particular antigen in your blood. For example, if you have typhoid, surface proteins of  the causative organism (Salmonella typhi) will act as antigen and when antibody (specific protein produced by your body against the antigen) is added to the infected blood sample antibody - antigen interaction occurs, which could be detected by various techniques.

Possible methods for diagnosing a disease!

There are several methods available for diagnosing a disease, but, most of them are based on the antigen-antibody interaction. Here I had listed a few methods of diagnosis, which I know:

1) ELISA  - Enzyme Linked Immunosorbent Assay!
This method is based on the antigen antibody interaction. Two antibodies are used for detecting the presence of a single antigen. But, both the antibodies won't be directly reacting with the antigen, one antibody (primary) will be reacting with the antigen, and other antibody(secondary -which was raised against the primary antibody) will react further with the primary antibody used, producing color based on the enzyme linked with it. Generally done using 96 wells plate.

2) Western Blot!
This is also done for finding the presence of a specific protein (in diagnosis, the protein is antigen) in a given sample.This is done generally after doing SDS PAGE and transferring the protein separated in the gel to nitrocellulose membrane followed by similar technique of adding primary and secondary antibody, which will show color if there is infection.

3) PCR - Polymerase Chain Reaction
Though the presence of a particular gene could be detected using Southern blot, the most sensitive technique used for diagnosing a disease based on genes is PCR. Quantitative PCR could be done for even finding the severity of the disease based on the concentration of a particular gene of the causative organism. 



I prefer to go with PCR than any other technique, but, when there is no thermo cycler in your laboratory it's better to go with ELISA! There are also other techniques like latex agglutination and microscopic observation of samples, but, the best way is PCR!

What do you say? PCR?

Wednesday, July 31, 2013

Possible mistakes while doing SDS PAGE!

Hi, dear readers, friends, it's been a long time, I wrote here. Sorry for that, was a bit busy in lab! And, you can be happy because of the fact that I was busy, as I got lots of experience to share with you! Let us first start with SDS PAGE!

SDS PAGE - Sodium Dodecyl sulphate - Poly Acrylamide Gel Electrophoresis! This SDS PAGE is done for separating proteins based on their molecular weight. It is a widely used technique and it is very useful for having an idea about the expression of your protein of interest.

Principle:
The name SDS PAGE comes from the fact that this method uses SDS for making your protein uniformly negatively charged and of course, the gel is prepared using poly acrylamide. Why to make the protein negatively charged? Because, here our interest is to separate proteins based only on their molecular weight, but not based on charge and all proteins are not negatively charged like DNA (which is separated based on size using Agarose Gel electrophoresis). SDS is an anionic detergent and it makes your protein negatively charged. Hence your protein moves towards the positive electrode. i.e. anode.

SDS PAGE 

Okay, the principle is fine, but, what are all the possible mistakes that one would do, while doing SDS PAGE? Here, I'll explain some possible mistakes, so that it would help beginners. I recommend you to learn the procedure for SDS PAGE before reading this.

While casting Gel!

  1. The first possible mistake you would do is, regarding the volume of gel that you should prepare! The plates - thick and thin plates. The thick plate comes in different thickness like 1mm, 1.5 mm etc., If you are preparing gel using 1.5mm you need different volume of gel and different volume for 1 mm. So, before preparing the gel, be clear about the volume that you need. If you prepare more, then obviously, its wastage of chemicals, if you prepare less, it is waste of chemicals even now, as your gel won't come up to the top of the plate to dip the comb in (then you must discard and prepare the right volume once again) 
  2. Place the plates right in the gel cast right, check that they are equal i.e not up and down. If you place one slightly up and fix it in the gel cast,  then your gel will leak out! 
  3. Check for leakage always with water before filling your gel in! Don't be over confident that your plates would never leak!
While loading your sample!
  1. Check you are loading your sample only inside the well and not into the tank, because there are possibilities for mis-loading, so double check before releasing your valuable protein sample! (it's valuable isn't it?)
  2. Remember to note down the order in which you load, because there is no point in running gel with out knowing the order of your samples in the gel! This might appear silly, but, happens!
While running!
  1. keep watching your gel, else all your sample would run out! oops, waste of time, chemicals and your hard work!
Wondering? how I'm listing lots of mistakes? Because, I had committed some of these mistakes and I had seen people commiting the rest! Making mistakes, is nothing wrong, but, repeating the same mistake is!

So, remember these things, save your time and chemicals! 

Thursday, June 20, 2013

Oral Cancer & HPV

Oral cancer refers to cancer in any part of your mouth like tongue, lips, palate etc., Cancer may be due to various reasons like irritation due to continuous alcohol consumption, smoking, poor oral hygiene etc., And it can also happen due to Human Papilloma Virus (HPV).

Human Papilloma Virus is a type of non enveloped virus (Papilloma) that can infect humans. They are classified generally as "High Risk" and "Low Risk" types. The high risk type HPV 16 is found to cause oro-pharyngeal cancer.

Diagnosis: Generally biopsy is done where a small part of your tumor is removed and observed under microscope. The biopsy can be just cutting a small portion or it can also be done using brush for collecting the cells.

After confirming the abnormality using this microscopic observation, further analysis is done for finding out the type of HPV which caused the oral cancer or to find whether the cancer is just because of alcohol/smoking.

This further analysis can be done by genotyping for exactly finding out the type of HPV. This genotyping could be done by several methods. Some of the methods of genotyping are

  1. Line Probe Assay
  2. Assay based on Melting Temperature
  3. Using micro-arrays
  4. Hybrid Capture Test
  5. Multiplex PCR
All these methods have their own advantages and disadvantages. Among them, the multiplex PCR is found to be very effective.

Symptoms: Any visible tumors or lesions which remains uncured more than two or three weeks. Difficulty in chewing, swallowing etc., This might also be the symptoms of other problems, but, it is better to go and consult a doctor when you have these symptoms.

To avoid oral cancer, avoid alcohol, smoking and maintain good oral hygiene.

Monday, June 10, 2013

First few days at RGCB as SRF!

It was 24th December I believe! I just saw the details of the Indian Academy of Fellowship only a week before the last date. I decided to apply for it and just applied in a hurry actually, yes, it was really a hurry! I was done with everything and sent the application.

I was waiting for the release of selected list of candidates from the first of March. They updated the selection list everyday but my name was not on the list till 25th of March! I used to go to the IAS website everyday and check the list, but, my name was not there in the selected list of candidates. I lost all my hope and decided that I won't get selected. But, it happened! I received a mail from the Academy after 25th of March! I was happy to the core, jumping up and down. Because, I was never out of my town from my child hood all alone. And, this fellowship was something really precious for me!

I believed that this would change my destiny to a better place! I was happy till my father said "no" to this. He was really afraid to send me out of my town! Yes, people in our town find it difficult to end their daughters for work or studies. But, I managed to convince my dad and he accepted to allow me to take up my fellowship.

And, the next problem after convincing my dad was, "when to join for the fellowship?" After lots of confusions, finally joined at Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram which was allotted as a centre for my fellowship on 4th June.

And, this is the first time for me, away from home! That too, in another state where I don't know the language. I got accommodation in the Centre's hostel. My room mate, she is a Dehradun and I'm really enjoying her company.

IAS
In the centre, I got placed in Cancer Research Department and now I'm working on Tongue cancer. Cancer Research became my dream when I gained interest in molecular biology. And now, my dream is getting true!

You'll feel great when your dream is coming true and I'm feeling it now, right now! For many people this might sound a bit exaggerated, but, really only I can feel how much this is important for me! And, really I can feel the value of this opportunity.

This research experience will surely help me to get into a University for my graduation. And to the people who want to join RGCB, this is an awesome place, you can very well join here for better research experience! I recommend RGCB for getting Summer training.

In RGCB they provide accommodation for the fellows inside the campus in their hostel which is useful for the students from other states/ places.

The normal lab timing is from 9.30 a.m to 5.30 p.m. And students do research even late nights which shows their ultimate interest. People here are dedicated!

Learning many things apart from the academics, which will be very useful for the rest of my life. It is very safe in here and the place is really "God's own Country". People here are very friendly, smiling and caring, so that getting acquainted with the new environment becomes very easy!

Finally, I'm feeling free and independent for the first time in my life with a complete satisfaction of what I'm doing right now!

Want the IAS fellowship? Read "this" and apply next year! My Best wishes!

[ I did write this post as it might motivate someone or can serve as a piece of information who want to know about the SRF experience at RGCB]

Genotyping, Phenotyping, Karyotyping!

We are going to discuss here about three different typing! They are genotyping, phenotyping and karyotyping. Before learning about them let us learn what is genotype, phenotype and karyotype!
Genotype, phenotype and karyotype

Genotype refers to the genetic make up of an organism. Generally the genes of an organism. The genotype of an organism can be represented as BB or Bb or bb based on the gene. If a person is having two recessive CFTR genes, then he will be getting cytic fibrosis. Genotype of an organism has also effect over the phenotype. Thus, genotype is representing the alleles of a gene in general. Genotyping is generally done based on PCR or hybridization.

Phenotype refers to the visible characters like structure, color and also the biochemical characters. This is based on the phenotype. Phenotyping can be done using biochemical assays.

Karyotype refers to the number of structure of chromosomes in an organism. karyotyping is done by staining and visualising the cells under microscope.

Friday, May 31, 2013

I'm in love!

As a biotechnology student whether I'm taking care of my meals plate or not, I have to take good care of my petriplates. I wash my petriplates better than my meals plate!

Yes, we never autoclave and sterilise our meals plate, but we do for our petriplates! We ( I mean me and my friends) enjoy cleaning and autoclaving our petriplates in our  department media room. And you know, my friends always complaint that I'm never accompanying them in cleaning. Yes, I'll go, wrap my petriplates, put it in autoclave, but I find it very hard to wash!

Always, I do autoclave, but, never wash after the autoclaving, I'll give that easy job to my friends :P I love my petriplates! Yes, I love them a lot.

But, how do I say that I love my petriplates? I love them because I'm always with them. I love them because, even whether I do complex Genetic engineering or simple Microbiology, I always need them!

And the final reason for loving my petriplates is they are so cute, especially, the glass ones! Wow, they are so cute!

And, today I loved them even more looking at the google doodle! Wow, Awesome it was.
For the 161st birthday of Julius Richard Petri - German microbiologist who invented petriplates, google had done this cool doodle. The six plates, each showing the source of the microbe when you place the mouse over it, looked really cool.

And, the best thing I loved was, Wikipedia has updated the information in the page of Julius Richard Petri adding, "google made a doodle for his 161st birthday!"

How fast, I had never noticed previously about this update of Wikipedia on google doodle, just today I noticed it and I was surprised! This is really really awesome. You didn't look at the doodle? Go, check it out, it is "sooper" :)

Love you Petri! <3 :) :P :)

Saturday, May 25, 2013

Types of plasmid!

I learnt genetic engineering, and of course, about plasmids. But, when I was questioned about the types of plasmid, God, I was blinking! :(

I had known plasmid only as a vector most times, I was aware of the types of vectors but, types of plasmids? :(

Okay, It's not a great shame, I learnt it now! Let me share here about the types of plasmids, just what I learnt.

Based on the functions of a plasmid they are classified as,

Resistance Plasmids:
These are the plasmids which have resistance genes. For example the gene coding beta lactamase which offers resistance against beta lactum antibodies like ampicillin. This is an important thing which is used while performing cloning experiments for checking the transformation. This was called as "R Factor" before the discovery of plasmids.

Fertility plasmids (F Plasmids):
And, these type of plasmids they have important genes called tra genes, never wonder, tra genes are transfer genes which are essential for the non sexual transfer in bacteria. This helps in formation of Pili and conjugation as a result.

Col plasmids:
These plasmids are the ones which have special genes for inhibiting the growth of similar bacterial strains i.e., bacteriocins.

Degenerative plasmids:
These are things which contains special genes responsible for digesting toulene like things. For example, I could say "Super bugs" they have engineered plasmid containing genes for digesting several types of chemicals and it is used for clearing oilspills.

Virulence plasmids:
These are the ones which makes us to hate bacteria. Yes, these make the bacteria virulent i.e., disease causing. You hate it, right?

Still I have one more thing to explain, "Promiscuous plasmid". This was asked in my university exam and adding to the sadder part, I didn't know the answer. But, nothing to worry, I got the answer now.

Promiscuous plasmids: These are plasmids which are self transmissible to a wide variety of organisms. They allow transfer of DNA to other species.

You know any other types of plasmids? Then share with me in the comment section, anything was not clear? comment then.

Take care. Bye :) :)

Tuesday, May 14, 2013

Modern Health care!

Everyone knows that India is developing in all the sectors and certainly, Medical sector is also one among them. And, just a few days back, higher secondary exam results were out and all the toppers were saying, "I would like to become a doctor and serve poor-and-needy". Most of the students want to become a doctor or an engineer. Okay, that's not our interest now.

There is no doubt that our country had already got very good hospitals and modern facilities. I had read in some articles that even people are coming from other countries to get treatment in India and I'm proud of it!

It is possible to modify the genes of your baby even when he/she is in the womb. You don't have a hormone, your body failed to secrete a hormone? Don't worry, just get a dose of it in the form of injection and be cool and healthy. I'm a biotechnology student, I'm damn passionate and interested in cancer research and you know? nowadays many people are coming out with new therapies and techniques for treating deadly diseases like cancer, not only cancer! Even magnets are used for treating cancer!

Is it possible to produce banana or apple which contains Insulin? Yes it's possible, by just expressing the human insulin gene in fruits of Banana plant. How cool it is, you can get a banana and enjoy it if you have diabetes! (This is just an example, don't go much deep and argue with me that banana is not good for Diabetic people, then, have some other fruit :) )These are all some innovations and modern things. These are all very few of the Biotechnology based innovative things that I know.

And there are also lots of innovations in the field of Bio-medical engineering which provides advanced equipment for the doctors to touch the lives of people in a better way!

Everything seems fine, right? Yes, all the techniques and technology are fine. But how far it is reaching the people? How far people are benefited? There is no doubt that many people are getting benefited by these techniques and technology. But, who are those "many"?

Modern Health Care
Unfortunately those "many" doesn't include the poor people! We have advanced technologies, but, they are very costly! I'm not blaming anybody here, but, this is the reality, isn't it? I know many people who are seeking other's help for paying their surgeries and medicines. Here we can't blame the hospitals for giving expensive treatments for their patients, we can't, because the technologies, the advanced technologies are costly! Then we can blame our people for being poor? I don't know that! So, here whether I can conclude saying "Modern health care is touching only the lives of rich, richer and richest people of our country?"


There are medicines available for most of the diseases in our country. But, whether we are all aware about the fact that we are all resistant to those medicines? Yes, even if you take antibiotic for your fever, it won't work anymore! Reason? Any medicine or antibiotic must be taken for the appropriate period of time as per the doctor's prescription. If doctor asks you to take medicine for one week, then you must do for a week. But, most of us won't do it. If we are feeling alright by the 3rd day, we'll stop the medicines and if your microbe is still present in very minute less quantity, then it will develop resistance against the antibiotic. Then after this, even if you take medicine already available for this fever again, you won't get better! (I hope. i explained it clearly in a simple way) This is why our Government is trying to create awareness in the case of Tuberculosis treatment.

There are many people in our country who are not educated, whether they are all aware of these things? No? Then, what's the use of having such innovative medicines and techniques? Then, here, whether I can conclude saying, "Modern health care is touching only the lives of literates in our country?"

We have all the facilities in our country, but, it is reaching all the people?  I leave it to you, you can think and say me the answer!
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Thanks to Indiblogger and Apollo hospitals who made me write this for "the contest". I had written previously about the drug resistance, but, I feel this will reach many than my previous one.

And finally, I'm afraid that I wrote something against somebody! :P Who cares, what I wrote here is, what I felt and what I know! :D :) Cheers!

Wednesday, May 8, 2013

Drinking chlorophyll!


Chlorophyll. I know this as a green pigment in plants and some algae and bacteria, which helps in photosynthesis. This is the thing, known by most people who know the basic botany! But, recently I got to know something interesting about this greeny pigment.

Few days back, I went to an exhibition in my town where I had come across a stall which had some plant based products and medicines. There I came across a product which was a "chlorophyll drink". I don't know why that thing alone got my interest among the number of products.

The lady who was selling those products came near me looking at my onterest over that product and she started explaining "this is good for diabetes, good for cancer, ulcer,...", she possibly listed out all the diseases she knows and added that she had cured cancer patients in the severe stages using this chlorophyll drink. And when she was explaining I thought like "yeah. we are consuming chlorophyll every day in the form of greeny spinach leaves..., and ofcourse having spinach is good for health"

I wondered! Just a drink could cure all the diseases??! Then I started searching about the health benefits of chlorophyll.

Chemically similar!
When we look at the chemical sturcture of chlorophyll and hemoglobin,  both resembles similar! Chlorophyll contains Mg(2+) in the centre and hemoglobin has Fe(2+) in the middle. So, drinking chlorophyll will surely give some benefits, isn't it?

Anti cancerous!
Certain cancers caused due to fungus present in contaminated foods are reduced by consuming chlorophyll! Chlorophyll attach to certain chemical components which causes cancer thereby blocking them.

Anti diabetic!
Ligands for the retinoid X receptor(RXR) had shown anti diabetic effects. Phytanic acid, which is a metabolite of chlorophyll is a natural ligand for RXR and hence it has anti diabetic effect.

Others:
I read several articles and papers regarding the benefits of chloropyll. I came across lots of benefits in having a chlorophyll diet. All the benefits are generally because of improving the immunity of the body.

But, Drinking chlorophyll is how far advisable? I feel like, there is no need for drinking chlorophyll. Because there are reports of side effects, drinking chlorophyll.

I remember one thing which my mom always stresses from my childhood, "Eat green veggies and spinach"

So, just have lots of greeens and get all the benefits like anticancerous, antidiabetic etc.,

Thursday, April 11, 2013

Be ready to fail, if you wanna be a Bio-technologist!

Fail??? "Oh, come on, you should not say like this", is this what you are feeling, after looking at this title? If so, don't worry, I'm not meaning this in a negative way as you think.

"Everything in life has ups and downs. What about this field of Biotechnology? It's not preferred by many students. Yes, I agree, it is not like the IT field, you can't find a perfect job after completing just your under-graduation, even if you find a job, that won't pay you much. So, obviously, people will go for areas which would give them a bagful of money.

"You think, Biotechnology is worthless then? you say, you won't get paid?", is this your next question?
If so, I'm not saying that.

Biotechnology, here, you can achieve heights only after years of enthusiasm and work in your laboratory. It's not the matter of earning money. This field is for people who want to achieve or discover something which is in your own body, in plants around you  in animals and in all living things around you.

And, now, we came away from our talk on failure. Why I mentioned like, be ready to fail. It's because, you won't succeed in most of your experiments in a single attempt! And, that's why I asked you to be ready.

When we try to clone in our lab, we repeated the ligation step twice, we tried different vectors even. And at last we succeeded, it's not like debugging a  C or C++ coding where you could look into the coding with your naked eye, you have to trouble shoot something which you can't see with your naked eyes! Looks difficult? But, it won't be, when you love this beautiful field - Biotechnology.

Failures will become a part of your life and you will enjoy your success which comes after every failure. And, the moment see your experiment giving positive result, ah, that is happiness, the real happiness. Only you would feel this, if you were a biology person! (or a person who has maximum interest in any field.

Every little thing you do in your lab will fail, but, don't worry, it'll be followed by a success!

Sunday, March 31, 2013

Southern Blotting / Hybridisation!

Southern blotting is used for finding specific DNA sequence from a DNA sample by using the combination of techniques like  gel electrophoresis, capillary electrophoresis and hybridization. The DNA from the gel (after gel electrophoresis)is transferred to a nitrocellulose membrane and hybridization analysis is done for finding out the sequence of our interest using a probe)

Let us discuss here the steps involved in doing Southern blotting. The steps are
1) Preparation of Sample
2) Restriction of the Sample
3) Gel Electrophoresis
4) Pre-treatment of the gel
5) Blotting
6) Hybridization

1) Preparation of Sample: (plant/animal/bacterial)

For tissue or blood samples the DNA could be obtained by treating with SDS (Sodium dodecyl Sulphate)
If we are going to have the DNA from a bacterial sample, there are separate protocols for extracting genomic DNA and pDNA. For extracting DNA from plants, CTAB (cetyltrimethyl ammonium bromide)
is used. This CTAB binds with the DNA and helps in better extraction using phenol. This CTAB is used specifically for plants, because they contain more carbohydrates than animal and bacterial cells.

2) Restriction of the Sample:

Specific restriction enzymes could be used fro restricting the sample of DNA.  Only one restriction enzyme can be used or more than one enzyme could also be used.  For restricting the sample of DNA containing 1 micro gram per microlitrethe following amount of buffer, enzyme could be used. (Choose the buffer corresponding to the enzyme you use) This volume could also be scaled up.

Components
Volume (micro litres)
10X buffer
1.0
DNA
1
Water
7.5
Enzyme
0.5
Total
10









3) Gel Electrophoresis: 
Agarose gel electrophoresis is the next step. The restricted sample is run in the agarose gel (% of the gel depends on the sample)

4) Pre-treatment of the gel:
The gel is pre-treated with 0.25 mol/litre HCl for 30 min and also treated with an alkali.
The reasons for pre-treatment:

  • Acid treatment helps in breaking the DNA in the bands formed in the gel into smaller fragments and this also leads to little depurination!
  • Alkali treatment is done for denaturing the DNA by breaking the "H" bonds. This helps the DNA in binding easily to the membrane during the blotting. And also this helps in the hybridization with the probe.
5) Capillary Blotting:

This could be done by using nitrocellulose membrane or nylon membrane.

i) Using nitrocellulose membrane (high salt transfer)

While using nitrocellulose membrane, gel must be neutralized after the alkali treatment because at the pH above 9, there won’t be efficient binding of DNA with the nitrocellulose membrane. The neutralization is done by soaking the gel in Tris – salt buffer.

Blotting is generally done using nitrocellulose membrane. The capillary electrophoresis is set up as shown in the figure using SSC buffer (20X) (SSC contains 3mol/litre Sodium chloride and 0.3 mol/litre Sodium citrate) blotting for about 18 hours.

After the blotting or transfer is over, the membrane is washed with SSC (2X) and then dried. In this case, the DNA won’t be firmly attached to the membrane, so, the membrane could be baked at 80degree C for 2 hours which will result in semi permanent attachment of the DNA to the membrane

This same procedure followed for the uncharged nylon membrane also, till the washing and drying. After that for strong binding of DNA with the membrane, the nylon membrane is UV treated.

ii) Using a positive charged nylon membrane:
In this method, a different buffer is used and no alkali pre treatment of the gel is needed. Here, O.4 mol/litre NaOH is used as buffer and this gives immediate covalent binding of the DNA to the membrane and hence no baking or UV treatment is needed.

Advantages of nylon membrane :
i)                    Better binding of DNA, even 50bp DNA binds efficiently whereas nitrocellulose membrane efficiently binds 500bp only.
ii)                   Nylon membranes cannot be damaged easily and hybridization could be done repeatedly upto 8 or 10 times.
iii)                 No need for post blotting baking or UV treatment for +ve charged nylon membranes and has reduced loss of DNA.

Electrophoresis or electroblotting from the gel to membrane could be done instead of capillary blotting and also vacuum blotting which draws the buffer through the gel to the membrane could also be done. This reduces the time of blotting to a few hours to minutes.

6)Hybridization:
Pre-hybridization step: This is done to block the areas of the membrane which donot contain DNA, to avoid random hybridization during hybridization. Generally this is done by treating the membrane in the buffer containing Salmon sperm DNA (popular choice of DNA used for blocking) for 15 minutes to 3hours depending on the membrane type.

Then actual hybridization is done by using high salt buffer with a detergent (2X SSC + 1% SDS) containing the probe DNA sequence. For increased sensitivity 10%Dextran sulphate or 8% polyethylene glycol 6000 is added to the buffer and probe mixture, this increases the cross linking between the probe and sample DNA. For increasing the specificity, the temperature at which the bonding between the probe and the sample DNA will be stable must be used; at this temperature all the other random bonding will be unstable.

Thus, we could identify the DNA fragment containing our interested sequence using Southern blotting.


Any queries? feel free to comment. 


Tuesday, March 19, 2013

Plant Bio Reactors (PBR)

You might be aware of using E.coli for producing human proteins. E.coli is a well established expression system and easy to handle. And many people are working on this E.coli (even I'm handling only E.coli in my laboratory most times)

Okay, we are fine with E.coli, and we are producing our protein of interest in that. And what is the need for an eukaryotic expression system? That thing, I explained in one of my previous posts (Clone human genes into plants!)

I explained in that post like we are expressing our human proteins and we are growing the entire plant for obtaining our protein of interest. And we need not do that all time and there is a choice of growing plant cells in bio reactors!

Yes, we do grow E.coli in bio reactors and why not the plant cells? You might be aware of this plant bio reactors and here I'm going to share something that I know about them. 


Plant bio reactors

I attended a seminar on "Advances in plant biotechnology" and as you guessed, this post is the result of that.
Okay, let me jump into the topic.

Important factors to be considered while designing a PBR are:
  • Plant cells (as well as animal cells) are shear sensitive
  • Plant cells form aggregates - and this aggregate formation sometimes helps you with your production
  • Risk of contamination

 I consider that these three things are very important in growing your plant cells in a PBR.

Shear sensitivity:
As, the plant cells are shear sensitive, we can't use the same reactor which we use for growing our microbes for growing plant cells. If you use a super fast impeller for effective mixing to eliminate the mass transfer limitations, then I'm sure your plant cells are gonna die. So, special design is needed for growing the plant cells.

So, there must be a balanced mixing such that the mass transfer is good and also the cells should
not lyse!

Aggregate formation:
Plant cells also form aggregates like bacterial cells. We don't prefer aggregate formation in case of bacterial cells. But, in plant cells, depending on the species, this aggregate formation could improve our productivity.
On the other hand, this aggregate formation causes mass transfer limitations thus affecting your productivity.

"???" You feel like "oh God"? And your question is "whether to have aggregates in plant cell reactors or not?"

Let me answer you. You need to have aggregates if it benefits you with your productivity but the same time, the mass transfer must also be good. For having the two things in balance, we have to maintain a "particular aggregate size", so that, our productivity will be fine in both the aspects like mass transfer is good and aggregate formation improving the productivity.

Then, the next question may be "how to maintain the optimum aggregate size?"

The optimum aggregate size could be maintained by using immobilization technique. You could immobilize your cells i.e. the optimum aggregate size would be maintained using immobilized plant cells.

Risk of contamination:
Comparing the bacterial cells, plant cells have a very high risk of contamination. Even when we grow them for producing callus, there is a lot of contamination risk especially fungal infection.

So, growing them in a reactor, that will be difficult job especially in case of maintaining the sterile environment. Most times, continuous reactors are not used for bacterial cultures as it has high risk of contamination comparing the batch and fed batch. I had never seen or done continuous culture of bacteria in our lab, normally we don't do continuous culture as it goes for more than a day and also it is difficult to maintain sterility. And, even when on of my seniors wanted to try continuous culturing, we were all saying "no you better try batch instead" fearing that everything may get spoiled due to contamination.

But, if high sterility is maintained, you can do continuous culturing of plant cells also.

Hope you understood what i explained, got doubts? Comment or e-mail, will answer you as soon as possible.


Sunday, March 3, 2013

Tuberculosis Diagnosis!

Hi, Today I'm going to share something about TB! TB - Tuberculosis is generally caused by Mycobacterium tuberculosis. The most common symptom of TB is "non -stop" cough!

They say, many people in India are "carrier" of the bacteria, but not the disease! Oh God! if you are in India, better go and check for this bacteria! The possibility of the spread of the disease could be due to unhygienic conditions!

You spit in public places? you sneeze? cough? No problem, you can sneeze or cough, but be careful! Careful coughing? careful sneezing? yes, you might spread the population this mycobacterium you are carrying (you need not have TB), so, be careful in sneezing and coughing!

Okay, let us come to the point, I started typing this with a view of giving an overview about "the diagnosis of Tuberculosis".

The most common test used for checking TB is "Tuberculin Skin Test". Let me explain you how this is done!

Tuberculin Skin Test!
Let us first look into the principle behind this test. When a person is infected with Mycobacterium tuberculosis, then, he will be carrying antibodies against it. When he/she is injected with the antigen from this bacteria, he will produce an induration (hard bulge in the place where the antigen is injected).  This could be found after sufficient time like 72 hours.This is used for diagnosing TB! In this case, false positive results are obtained when a person tested for the second time using this method.

Antigen? What is the antigen used here? Is that your question? It is the protein which triggers the immune response extracted from the mycobacterium sp.

 Do you think this is enough for diagnosing TB? "Okay, what else one could do for diagnosing TB?" When my professor asked this question in class, I replied "ELISA".

ELISA - Enzyme Linked Immuno Sorbent Assay: Here the blood sample from the patient must be collected and checked for antigen antibody interaction in the well plate. Here we must have the antibody available in our lab. If there is antigen antibody interaction the ELISA produces colour else it won't!

"Okay fine. What else you could do for diagnosing TB?", when my professor questioned further, I replied, "PCR"! He was happy atlast that I had answered as he expected!

PCR: How PCR could be done for diagnosing TB? Mycobacterium genome sequence is available online in the databases and one could design primers based on that and when PCR works, then the person is having TB, else not! Real Time PCR is generally preferred than the normal PCR for avoiding errors.

The advantage of PCR is that one could also say the severity of the disease and dosage of the antibiotic could be given accordingly.

After all, the dosage must be very correct and the antibiotic treatment should not be dropped in the middle to avoid development "multi drug resistant" species!

I had shared whatever I understood as an overview, any doubts? mistakes? suggestions? Feel free to comment!

Saturday, February 9, 2013

Clone human genes into plants! - Avidadham - International Conference on Molecular therapeutics!


E.coli VS Plants!

As an undergraduate of Biotechnology, I know about cloning. I know about cloning a gene which codes for a human protein into a bacterial cell, E.coli, most times.

I also know about producing transgenic plant varieties. But, I don't know about cloning a human gene into plant! I came to know about cloning a human gene into plant and producing the protein in plants from the workshop session of AVIDADHAM'13.

Let me explain something about this!

As you may know, eukaryotic gene has both introns and exons. So cloning a eukaryotic gene into a prokaryote will have lots of problem like splicing and post translational modification.

Generally for cloning eukaryotic gene into a prokaryote like E.coli, we do follow bottom up approach where we use mRNA to synthesise cDNA, to remove the noncoding introns. But, even if we remove the introns there will be problems with post translational modification.

So, to avoid this, we are cloning our gene from human into plants? Here, we are not going to grow the plant cells, but, the whole plant.

Plant tissue culture
I had come to know that they had successfully expressed the protein of interest in plants but the problem is with purifying and extracting the protein from the plants. Once this is possible, once the extraction procedures are found, once the extraction is done with out affecting the protein's activity, then, I'm sure, this is going to be a ground breaking technique.

We could get bulk amount of our protein. But there are lots of problems when we come to the ethical side, these GM plants should be grown only inside a green house, so that it won't affect the other wild varieties by pollination.

If we are growing this in open field then due to pollination there is a possibility that our wild varieties might become extinct. So, we must grow these plants inside the green house.

After all, if we come out with an idea saying "GM plants", there are people who are ready to boycott!

But, I got very much excited about this idea of cloning human genes into plants. It's awesome, isn't it?

Any queries? Feel free to comment! I'll try to answer my best!


Monday, January 21, 2013

How to clone a GENE?


Hi,

Wish you a very happy day! Let all you researches result only in success! J

You know, I’m learning cloning, wow, rhyming it is, learning – cloning!

Like this rhythm, cloning is also fun! Yes, fun, interesting! Let me share with you, what I know about cloning.

The first step of any cloning experiment would be selecting a vector. Now, I’m learning cloning using plasmid as a vector.

So, you have to choose a vector for cloning at first. Vector is nothing; it’s just a plasmid DNA into which we can incorporate our gene of interest and transform the vector into a host which will produce the protein coded by our gene of interest.

Vector need not be only p-DNA, viruses could also be used as vector. But, here, we are going to have an overview of cloning using Plasmid vector.

Okay, we had selected the vector, let us assume. What next? We have to look at our gene of interest now. We have to get the sequence of our gene of interest – it’s available in databases like NCBI.

Then, we have to isolate our gene of interest from the total genome of the organism. If it I prokaryote then you can just easily get the total genomic DNA and do a PCR (polymerase chain reaction) for amplifying your gene of interest.

If it is going to be an eukaryotic gene, then you must go in reverse. You must get the total RNA, then from that you have to isolate the mRNA, then from the mRNA you have to get your amplified gene of interest.

Before doing PCR, you must design Primer. The primer must have the restriction sequences at the end, so that the restriction sequence will also get inserted with your gene of interest at the ends.

Then after PCR, the gene f interest could be inserted into the vector just by restricting both your gene of interest and vector. They both will have complementary cohesive ends, so that they could hybridize with each other.

Of course, here there will be nick, but, we need not worry about the nick, once we transform this vector with gene of interest into our host – say E.Coli DH5 alpha, the host mechanism will take care of the nick.

E.coli DH5 alpha is a special strain of E.coli in which the defense mechanism against the foreign gene is knocked out, so that we could transform our gene into it and ask the E.coli to produce our protein, with out any problem of defense action.
This is just an overview of Cloning. There are several things to explain than this.

I’m happy that I know how to clone now! 

Happy cloning!!!

Saturday, January 5, 2013

Precise DNA cut!

We all know that we can cut and engineer our DNA or DNA of any other organism. But, so far the methods used for DNA cutting are not precise.

Available techniques:
By the older method, the genes are added into the cell and they get inserted into the genome of the cell in a random manner. This can’t be used when our aim is to engineer the DNA at a specific site and here we can’t also replace the already existing gene with our gene.

There is homologous recombination method, but unfortunately natural recombination won’t occur in cells that easily!

There are also methods which use Zinc fingers for delivering nucleases which cuts the DNA, but, this is really hard, because nuclease can’t target every possible DNA sequence. This is costly too.

TALENs – Transcription Activator Like Effector Nucleases – cut the DNA at specific location. But, what to do, this is also costly!

Our new method
Here, we are going to use the already  existing proteins of bacteria and  RNA. The tool contains RNA sequence with the nuclease called cas9.

Here, RNA is used for recognising the site on the genome where we have to make a cut and Cas9 for cutting! The Cas9 get activated once the RNA finds exact match. It won’t get activated, even if there is a single residue difference.

If we are willing to replace the already existing sequence with new one, we have to provide a DNA template.

This is not costly, yet effective and precise!

This is a good news for all Genetic Engineers, right? Even, I’m happy about this as a GE student, then for sure this is going to be a boon for Genetic Engineers.

After all, as my professor said, Playing with genes is the real fun! J

Aspartame - Artificial Sweetener!

We all love sweets, isn’t it? From a cute baby who had just started feeling and enjoying the tastes to 100 year old people, love sweets! Oh, I could hear you, yes, Diabetic patients can’t enjoy sweets. But, they too love to have sweets. Even, my father is diabetic and he uses to eat sweets hiding from my mom!

Okay, what for that sweet love, you are asking? Nothing wrong in that, but, if you are crazy about the bottled drinks, then, I’m sorry, you must start worrying! L

Aspartame – is an artificial sweetener which is used in most of the bottled drinks. And, that is an approved artificial sweetener by FDA (US Food and Drug Administration – which tests and approves all food and drug items before they are marketed)

“It is approved by FDA, then, why we must worry?”, is this your next question? Let me answer you!

Aspartame is formed using two aminoacids – L-Phenyl alanine and L aspartate. This when broken down during metabolism in our body produces – phenylalanine and hence must be avoided by people who are suffering from the genetic disorder called Phenyl Ketone Urea (PKU) – these people can’t metabolise phenyl alanine further!

This Aspartame will be unstable at higher temperatures and they are stable in the pH of 4 in general. As most of the bottled drinks have the pH of 3 to 5, this is generally used in bottled drinks.

Moreover, this is preferred because this has 200% greater sweetness than the normal sucrose we use. This is enough for us right? The taste? The taste is enough, right?

Okay, what is the problem in consuming this aspartame? Only the PKU people must avoid this?

No, even normal healthy individual’s health will be affected on consuming aspartame in long run – many researches revealed this!

There are several published articles narrating the link between the aspartame consumption and Cancer! Yes, cancer, leukemia!

In 1965 Aspartame was discovered and was approved by FDA initially in 1974. Even after the approval, the safety of aspartame is being questioned by many researchers and the FDA considered the approval again!

A trick to hide the risks behind a product is generally done by showing the results of short term tests and analysis. People are misled by such short term analysis!

The metabolized products of aspartame not only include aspartate and phenyl alanine but also methanol. Methanol is toxic! But, many scientists and researches argue that methanol could be degraded by alcohol dehydrogenases. But, this degradation results in formaldehyde which will degrade DNA and proteins! This will lead to several Auto immune diseases and cancer!

And, researches reveal that men are affected more by the consumption of aspartame!
 
Many products like this, which are synthesized artificially might have long term effects over our health, as this is going to affect us only in long run we won’t realize this.

My mom use to advice me not to take bottled drinks and she suggests fresh juices instead. She says, if I drink bottled items, it would affect my health.

It’s true isn’t it? Aspartame or other artificial sweeteners might be the reason behind this?

I’m not sure about the worst effects of Aspartame, I just wrote this post by reading various articles and information already published.

You are sick? May be it is due to artificial sweeteners? To check this, just avoid artificial sweeteners. Check the label of everything you consume sweet! If you are fine after avoiding these artificial sweets, then, the problem is with the artificial sweeteners.

 Just try to be safe with natural sweets!