Creating the clone

Creating the clone

The steps in cloning are as follows. DNA is extracted from the organism under study and is cut into small fragments of a size suitable for cloning. Most often this is achieved by cleaving the DNA with a restriction enzyme. Restriction enzymes are extracted from several different speciesand strains of bacteria, in which they act as defense mechanisms against viruses. They can be thought of as “molecular scissors,” cutting the DNA at specific target sequences. The most useful restriction enzymes make staggered cuts; that is, they leave a single-stranded overhang at the site of cleavage. These overhangs are very useful in cloning because the unpaired nucleotides will pair with other overhangs made using the same restriction enzyme. So, if the donor DNA and the vector DNA are both cut with the same enzyme, there is a strong possibility that the donor fragments and the cut vector will splice together because of the complementary overhangs. The resulting molecule is called recombinant DNA. It is recombinant in the sense that it is composed of DNA from two different sources. Thus, it is a type of DNA that would be impossible naturally and is an artifact created by DNA technology.

The next step in the cloning process is to cut the vector with the same restriction enzyme used to cut the donor DNA. Vectors have target sites for many different restriction enzymes, but the most convenient ones are those that occur only once in the vector molecule. This is because the restriction enzyme then merely opens up the vector ring, creating a space for the insertion of the donor DNA segment. Cut vector DNA and donor DNA are mixed in a test tube, and the complementary ends of both types of DNA unite randomly. Of course, several types of unions are possible: donor fragment to donor fragment, vector fragment to vector fragment, and, most important, vector fragment to donor fragment, which can be selected for. Recombinant DNA associations form spontaneously in the above manner, but these associations are not stable because, although the ends are paired, the sugar-phosphatebackbone of the DNA has not been sealed. This is accomplished by the application of an enzyme called DNA ligase, which seals the two segments, forming a continuous and stable double helix.

The mixture should now contain a population of vectors each containing a different donor insert. This solution is mixed with live bacterial cells that have been specially treated to make their cells more permeable to DNA. Recombinant molecules enter living cells in a process called transformation. Usually, only a single recombinant molecule will enter any individual bacterial cell. Once inside, the recombinant DNA molecule replicates like any other plasmid DNA molecule, and many copies are subsequently produced. Furthermore, when the bacterial cell divides, all of the daughter cells receive the recombinant plasmid, which again replicates in each daughter cell.

The original mixture of transformed bacterial cells is spread out on the surface of a growth medium in a flat dish (Petri dish) so that the cells are separated from one another. These individual cells are invisible to the naked eye, but as each cell undergoes successive rounds of cell division, visible colonies form. Each colony is a cell clone, but it is also a DNA clone because the recombinant vector has now been amplified by replication during every round of cell division. Thus, the Petri dish, which may contain many hundreds of distinct colonies, represents a large number of clones of different DNA fragments. This collection of clones is called a DNA library. By considering the size of the donor genome and the average size of the inserts in the recombinant DNA molecule, a researcher can calculate the number of clones needed to encompassthe entire donor genome, or, in other words, the number of clones needed to constitute a genomic library.

A genomic DNA library is a collection of DNA fragments that make up the full-length genome of an organism. A genomic library is created by isolating DNA from cells and then amplifying it using DNA cloning technology.

Another type of library is a cDNA library. Creation of a cDNA library begins with messenger ribonucleic acid (mRNA) instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA. The resulting DNA molecules are called complementary DNA (cDNA). A cDNA library represents a sampling of the transcribed genes, whereas a genomic library includes untranscribed regions.

A cDNA library represents a collection of only the genes that are encoded into proteins by an organism. Complementary DNA, or cDNA, is created through reverse transcription of messenger RNA, and a library of cDNAs is generated using DNA cloning technology.

Both genomic and cDNA libraries are made without regard to obtaining functional cloned donor fragments. Genomic clones do not necessarily contain full-length copies of genes. Furthermore, genomic DNA from eukaryotes (cells or organisms that have a nucleus) contains introns, which are regions of DNA that are not translated into protein and cannot be processed by bacterial cells. This means that even full-sized genes are not translated in their entirety. In addition, eukaryotic regulatory signals are different from those used by prokaryotes (cells or organisms lacking internal membranes—i.e., bacteria). However, it is possible to produce expression libraries by slicing cDNA inserts immediately adjacent to a bacterial promoter region on the vector; in these expression libraries, eukaryotic proteins are made in bacterial cells, which allows several important technological applications that are discussed below in DNA sequencing.

Several bacterial viruses have also been used as vectors. The most commonly used is the lambda phage. The central part of the lambda genome is not essential for the virus to replicate in Escherichia coli, so this can be excised using an appropriate restriction enzyme, and inserts from donor DNA can be spliced into the gap. In fact, when the phage repackages DNA into its protein capsule, it includes only DNA fragments the same length of the normal phage genome.

Vectors are chosen depending on the total amount of DNA that must be included in a library. Cosmids are engineered vectors that are hybrids of plasmid and phage lambda; however, they can carry larger inserts than either pUC plasmids (plasmids engineered to produce a very high number of DNA copies but that can accommodate only small inserts) or lambda phage alone. Bacterial artificial chromosomes (BACs) are vectors based on F-factor (fertility factor) plasmids of E. coli and can carry much larger amounts of DNA. Yeast artificial chromosomes (YACs) are vectors based on autonomously replicating plasmids of Saccharomyces cerevisiae (baker’s yeast). In yeast (a eukaryotic organism) a YAC behaves like a yeast chromosome and segregates properly into daughter cells. These vectors can carry the largest inserts of all and are used extensively in cloning large genomes such as the human genome.

NEXT Isolating the clone

What is DNA Cloning

DNA Cloning

In biology a clone is a group of individual cells or organisms descended from one progenitor. This means that the members of a clone are genetically identical, because cell replication produces identical daughter cells each time. The use of the word clone has been extended to recombinant DNA technology, which has provided scientists with the ability to produce many copies of a single fragment of DNA, such as a gene, creating identical copies that constitute a DNA clone. In practice the procedure is carried out by inserting a DNA fragment into a small DNA molecule and then allowing this molecule to replicate inside a simple living cell such as a bacterium. The small replicating molecule is called a DNA vector (carrier). The most commonly used vectors are plasmids (circular DNA molecules that originated from bacteria), viruses, and yeast cells. Plasmids are not a part of the main cellular genome, but they can carry genes that provide the host cell with useful properties, such as drug resistance, mating ability, and toxin production. They are small enough to be conveniently manipulated experimentally, and, furthermore, they will carry extra DNA that is spliced into them.

Steps involved in the engineering of a recombinant DNA molecule.

NEXT CREATING THE CLONE

Recombinant DNA technology

Recombinant DNA technology, joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. Since the focus of all genetics is the gene, the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes. Although it is relatively easy to isolate a sample of DNA from a collection of cells, finding a specific gene within this DNA sample can be compared to finding a needle in a haystack. Consider the fact that each human cell contains approximately 2 metres (6 feet) of DNA. Therefore, a small tissue sample will contain many kilometres of DNA. However, recombinant DNA technology has made it possible to isolate one gene or any other segment of DNA, enabling researchers to determine its nucleotide sequence, study its transcripts, mutate it in highly specific ways, and reinsert the modified sequence into a living organism.

The process of DNA extraction is necessary to isolate molecules of DNA from cells or tissues. A series of steps, including the use of protease enzymes to strip proteins from the DNA, are required for isolating pure DNA that is suitable for use in later procedures, such as cloning or sequencing.

Fermentation and anaerobic respiration

Introduction

Ever wonder how yeast ferment barley malt into beer? Or how your muscles keep working when you're exercising so hard that they're very low on oxygen?

Both of these processes can happen thanks to alternative glucose breakdown pathways that occur when normal, oxygen-using (aerobic) cellular respiration is not possible—that is, when oxygen isn't around to act as an acceptor at the end of the electron transport chain. These fermentation pathways consist of glycolysis with some extra reactions tacked on at the end. In yeast, the extra reactions make alcohol, while in your muscles, they make lactic acid.

Fermentation is a widespread pathway, but it is not the only way to get energy from fuels anaerobically (in the absence of oxygen). Some living systems instead use an inorganic molecule other than \text {O}_2O2​O, start subscript, 2, end subscript, such as sulfate, as a final electron acceptor for an electron transport chain. This process, called anaerobic cellular respiration, is performed by some bacteria and archaea.

In this article, we'll take a closer look at anaerobic cellular respiration and at the different types of fermentation.

Anaerobic cellular respiration

Anaerobic cellular respiration is similar to aerobic cellular respiration in that electrons extracted from a fuel molecule are passed through an electron transport chain, driving \text{ATP}ATPA, T, P synthesis. Some organisms use sulfate (\text {SO}_4^{2-})(SO42−​)left parenthesis, S, O, start subscript, 4, end subscript, start superscript, 2, minus, end superscript, right parenthesis as the final electron acceptor at the end ot the transport chain, while others use nitrate (\text {NO}_{3}^-)(NO3−​)left parenthesis, N, O, start subscript, 3, end subscript, start superscript, minus, end superscript, right parenthesis, sulfur, or one of a variety of other molecules^11start superscript, 1, end superscript.

What kinds of organisms use anaerobic cellular respiration? Some prokaryotes—bacteria and archaea—that live in low-oxygen environments rely on anaerobic respiration to break down fuels. For example, some archaea called methanogens can use carbon dioxide as a terminal electron acceptor, producing methane as a by-product. Methanogens are found in soil and in the digestive systems of ruminants, a group of animals including cows and sheep.

Similarly, sulfate-reducing bacteria and Archaea use sulfate as a terminal electron acceptor, producing hydrogen sulfide (\text H_2\text S)(H2​S)left parenthesis, H, start subscript, 2, end subscript, S, right parenthesis as a byproduct. The image below is an aerial photograph of coastal waters, and the green patches indicate an overgrowth of sulfate-reducing bacteria.

Aerial photograph of coastal waters with blooms of sulfate-reducing bacteria appearing as large patches of green in the water.

Fermentation

Fermentation is another anaerobic (non-oxygen-requiring) pathway for breaking down glucose, one that's performed by many types of organisms and cells. In fermentation, the only energy extraction pathway is glycolysis, with one or two extra reactions tacked on at the end.

Fermentation and cellular respiration begin the same way, with glycolysis. In fermentation, however, the pyruvate made in glycolysis does not continue through oxidation and the citric acid cycle, and the electron transport chain does not run. Because the electron transport chain isn't functional, the \text{NADH}NADHN, A, D, Hmade in glycolysis cannot drop its electrons off there to turn back into \text {NAD}^+NAD+N, A, D, start superscript, plus, end superscript

The purpose of the extra reactions in fermentation, then, is to regenerate the electron carrier \text{NAD}^+NAD+N, A, D, start superscript, plus, end superscript from the \text{NADH}NADHN, A, D, H produced in glycolysis. The extra reactions accomplish this by letting \text{NADH}NADHN, A, D, H drop its electrons off with an organic molecule (such as pyruvate, the end product of glycolysis). This drop-off allows glycolysis to keep running by ensuring a steady supply of \text{NAD}^+NAD+N, A, D, start superscript, plus, end superscript.

Lactic acid fermentation

In lactic acid fermentation, \text{NADH}NADHN, A, D, H transfers its electrons directly to pyruvate, generating lactate as a byproduct. Lactate, which is just the deprotonated form of lactic acid, gives the process its name. The bacteria that make yogurt carry out lactic acid fermentation, as do the red blood cells in your body, which don’t have mitochondria and thus can’t perform cellular respiration.

Diagram of lactic acid fermentation. Lactic acid fermentation has two steps: glycolysis and NADH regeneration.

During glycolysis, one glucose molecule is converted to two pyruvate molecules, producing two net ATP and two NADH.

During NADH regeneration, the two NADH donate electrons and hydrogen atoms to the two pyruvate molecules, producing two lactate molecules and regenerating NAD+.

Muscle cells also carry out lactic acid fermentation, though only when they have too little oxygen for aerobic respiration to continue—for instance, when you’ve been exercising very hard. It was once thought that the accumulation of lactate in muscles was responsible for soreness caused by exercise, but recent research suggests this is probably not the case.

Lactic acid produced in muscle cells is transported through the bloodstream to the liver, where it’s converted back to pyruvate and processed normally in the remaining reactions of cellular respiration.

Alcohol fermentation

Another familiar fermentation process is alcohol fermentation, in which \text{NADH}NADHN, A, D, Hdonates its electrons to a derivative of pyruvate, producing ethanol.

Going from pyruvate to ethanol is a two-step process. In the first step, a carboxyl group is removed from pyruvate and released in as carbon dioxide, producing a two-carbon molecule called acetaldehyde. In the second step, \text{NADH}NADHN, A, D, H passes its electrons to acetaldehyde, regenerating \text{NAD}^+NAD+N, A, D, start superscript, plus, end superscript and forming ethanol.

Diagram of alcohol fermentation. Alcohol fermentation has two steps: glycolysis and NADH regeneration.

During glycolysis, one glucose molecule is converted to two pyruvate molecules, producing two net ATP and two NADH.

During NADH regeneration, the two pyruvate molecules are first converted to two acetaldehyde molecules, releasing two carbon dioxide molecules in the process. The two NADH then donate electrons and hydrogen atoms to the two pyruvate molecules, producing two lactate molecules and regenerating NAD+.

Alcohol fermentation by yeast produces the ethanol found in alcoholic drinks like beer and wine. However, alcohol is toxic to yeasts in large quantities (just as it is to humans), which puts an upper limit on the percentage alcohol in these drinks. Ethanol tolerance of yeast ranges from about 555 percent to 212121percent, depending on the yeast strain and environmental conditions.

Facultative and obligate anaerobes

Many bacteria and archaea are facultative anaerobes, meaning they can switch between aerobic respiration and anaerobic pathways (fermentation or anaerobic respiration) depending on the availability of oxygen. This approach allows lets them get more ATP out of their glucose molecules when oxygen is around—since aerobic cellular respiration makes more ATP than anaerobic pathways—but to keep metabolizing and stay alive when oxygen is scarce.

Other bacteria and archaea are obligate anaerobes, meaning they can live and grow only in the absence of oxygen. Oxygen is toxic to these microorganisms and injures or kills them on exposure. For instance, the Clostridium bacteria that are responsible for botulism (a form of food poisoning) are obligate anaerobes. Recently, some multicellular animals have even been discovered in deep-sea sediments that are free of oxygen.

Why the Sky is blue ?

The daytime sky on Earth appears blue in colour. Light is made up of PHOTONS which are matterless particles. White light is made up of seven colours: red, orange, yellow, green, blue, violet & indigo.

This light travels at different wavelengths. Some colours are better at passing through gases than others. Red light is seen at longer wavelengths and indigo has the shortest wavelength.

When photons from the Sun travel into Earth's atmosphere some of this light hits and bounces the nitrogen and oxygen molecules there. The result is that light is split up and scattered.

Red, orange, yellow and green light carries straight on through this atmosphere but blue, violet and indigo get bounced around from molecule to molecule.

We see a blue sky as a result of all this bouncing around.

Why don't we see an Indigo or Violet sky? Simply because our eyes do not see these colours very well. Blue is a more dominant colour.

Further evidence of this can be seen during sunset and sunrise. The sky appears red then as the light travels through more of Earth's atmosphere. Most of the blue light is scattered away. Also during a lunar eclipse the Moon appears red as light travels through Earth's atmosphere.

How towrite a Cover Letter?

Purpose of Your Cover Letter

Your cover letter is an important component of the application process. It serves as a way for you to summarize your qualifications, state your interest in a position, and stand out from other applicants. It is specific to each opportunity you are pursuing.

Cover letters should be well written and always accompanied by each resume you send out unless otherwise specified. It is particularly important to include a cover letter, if an objective is not listed on your resume, to be clear on what position you are interested in. 

Tips for Writing Your Cover Letter

Writing a cover letter can be overwhelming. Focus your efforts and include content that is concise, relevant, and appealing to potential employers.

Be purposeful

• While every cover letter is different, effective cover letters demonstrate you are a good fit for the position.
• Convey your enthusiasm for the position and knowledge of the company.
• Provide support and examples that showcase the skills and competencies that are being sought.
• Focus on your accomplishments and measurable results.

Follow standard business writing protocol 

• Address your cover letter to a specific person whenever possible. It may take some resourcefulness on your part to identify the appropriate person, but the letter will be better received.  
• Write clearly and concisely.
• Use proper grammar and check for misspelled words.
• Limit your letter to one page.
• Be sure to include the date, an appropriate salutation, and closure with your signature.

Do not mass produce 

• Mass produced cover letters are easy to detect. Be sure to relate your specific skills and experiences to each indivdual position.   
• Incorporate information that reflects your knowledge of the company, the industry, or the position. 
• Consider that employers are seeking to fill specific roles and are looking for applicants that have the skills and qualities to succeed in that role. 

Structuring Your Cover Letter

Follow these guidelines to ensure your cover letter is properly structured.  

Paragraph 1: Capture Attention 

• In your first paragraph, capture the reader's attention.
• Indicate the position you are applying for and how you learned of the vacancy, i.e. Did someone tell you about it?  Did you see an ad or website? 
• Outline the specific reasons why you are ideal for the position.  
• Sell yourself in paragraph 1. Do not wait until the second paragraph to articulate why you are well qualified for the position.

Paragraph 2 & 3: Create Desire 

• Describe yourself as a serious candidate and one worth inviting for an interview. State the hard details including your specific skills, history of responsibility, success, etc. 
• Think about ways to reinforce an image of yourself that includes as many of the desired qualities as possible. 
• Show, don’t tell. Remember, your goal is to set yourself apart from other applicants. Do not just tell the employer you have a skill, provide evidence. For example, do not just state you are “detail oriented”. Give the reader an example of something in your work history that proves that you are detail oriented. 
• Refer to your resume, but do not simply list the contents of it. 
• Emphasize how your variety of experiences are connected to the position and will benefit the company. 

Paragraph 4: Call for Action 

• Use a few lines to express your strong interest in the position and your desire to discuss your application further in an interview. 

Give a brief summary of the key points in the letter, but avoid repetition.

How to gain weight?

We often talk about losing weight, but this is what you need to consider if gaining weight is one of your health goals. 

For healthy weight gain you need to increase your calorie intake by increasing nutrient and calorie rich foods in the diet. You need to supply your body with all the essential nutrients it needs for muscle growth and maintenance. 

You should be aiming to put on healthy weight by increasing muscle mass with a healthy proportion of body fat. To do this you need to increase your daily food intake of high-protein foods, healthy fats and slow releasing complex carbohydrates. Steer away from heavily processed junk foods that contain 'empty calories' and are high in hydrogenated oils and sugar.

Protein is essential for growth and maintenance of muscles, healthy choices include oily fish, chicken, red meat, dairy foods and nuts and seeds. Protein powders are very useful for people trying to increase their weight, try adding some to smoothies.

Increasing your intake of complex carbohydrate foods such as wholegrain breads, pasta, oats and rice, and starchy vegetables (potato, sweet potato, pumpkin, corn), will help promote healthy weight gain, without disrupting your blood sugar levels.

Upping healthy unsaturated fats, replacing saturated and trans-fats, is also beneficial. Use more cold pressed oils (olive, macadamia, walnut and chia), nuts and seed pastes, and avocado. Try making salad dressings with cold pressed oils and tahini, satay sauces with natural peanut butter, pesto through pasta, and cheese sauces over vegetables. Fresh coconut flesh is a delicious addition to smoothies.

Try including more calorie and nutrient rich snacks in your daily diet such as nut butters, tahini or avocado on wholegrain toast; nuts and seeds with dried fruit; muesli bars (try making your own healthy ones); cheese, avocado and tomato on whole wheat crackers; or smoothies with protein powder, milk, LSA (ground linseeds, sunflower seeds and almonds), and banana.

Remember to eat plenty of fruits and vegetables as they should be a major part of any healthy diet. 

Conditions such as an over active thyroid can make it difficult for people to put on weight, and should be investigated and treated.