Biotechnology: Principles and Processes — Class 12 Biology Notes & Practice

Let us start with the honest bit. The first time most students open this chapter, the page looks like it was written in a different language. Restriction endonuclease. Palindromic nucleotide sequence. Insertional inactivation. Sparged stirred-tank bioreactor. It is a wall of long words, and the very natural reaction is to close the book and decide that biotechnology is for other people. If that is roughly where you are sitting right now, take a breath, because I have some good news for you: this is one of the friendliest chapters in the whole of Class 12 Biology once somebody walks you through it slowly. There is almost nothing to memorise blindly. There is one story, told in order, and every long word is just a label stuck onto a very ordinary idea.

Here is the story in one sentence. We want a bacterium to make a human protein for us. To do that we have to take a human gene out of a human cell, cut it out cleanly, make lots of copies of it, glue it into a little circular piece of bacterial DNA, push that circle into a bacterium, grow billions of those bacteria in a big steel tank, and then clean up the protein they make. That is the entire chapter. Seven steps. Everything else in these notes is simply a closer look at one of those seven steps, plus the tools you need to carry each one out.

These notes are built for the student who is starting from zero and for the student who has read the chapter three times and still cannot answer a question in the exam. We will build every idea from the ground up, with everyday comparisons that actually stick. You will get the full set of restriction enzymes class 12 notes you need, a properly laid out set of recombinant DNA technology steps with diagram, and at the end a worksheet of biotechnology principles and processes class 12 important questions with complete model answers and their mark splits, so you can see exactly what an examiner is hoping to read.

One quick note on where this sits in your syllabus. In the CBSE Class 12 Biology course for 2026-27, this chapter lives inside Unit IX, and that unit carries 12 marks in the theory paper. The syllabus line for this particular chapter is short and blunt: Genetic Engineering, or Recombinant DNA Technology. That single line is the umbrella under which the whole of the NCERT chapter sits, so we will teach all of it. Do not let anybody tell you that a short syllabus line means a small chapter.

Before you dive in, it is worth knowing that this chapter leans hard on things you already met earlier in the year. If the words double helix, base pairing, replication and transcription feel shaky, spend twenty minutes with our Molecular Basis of Inheritance chapter notes first. Everything here rests on that foundation. And once you finish this chapter, the natural next stop is the applications chapter, where you find out what all this machinery is actually used for: insulin, Bt cotton, gene therapy and more.

Take it one section at a time. You do not have to understand everything today. You only have to understand one more thing than you did yesterday.

What You’ll Learn

Your Game Plan

If you only have a few study sessions before your test, work through this chapter in this order. It is deliberately arranged so that nothing arrives before you are ready for it.

1. Get the big picture first. Read the seven-step workflow diagram near the end of these notes before you read anything else. Five minutes there will save you an hour of confusion later, because you will know where each new tool fits.
2. Learn the three tools. Restriction enzymes, vectors, and competent host cells. These are the only three things you need before the process makes sense. Give them one full session.
3. Walk the seven steps in order. Isolate, cut, separate, amplify, insert, grow, purify. Say them out loud until the order is automatic. Examiners love asking you to arrange them.
4. Draw, do not read. Redraw the pBR322 map and the PCR cycle from memory on blank paper. If you can draw it, you know it. If you can only recognise it, you do not.
5. Attack the worked examples. Each example card in these notes is written like a real exam question, with the mark split shown. Cover the answer, attempt it, then compare.
6. Finish with the worksheet. Ten questions at the end. Do them closed-book, then mark yourself honestly against the model answers.

Study Notes

Everything below is arranged so that each idea only depends on ideas you have already met. Read in order the first time. After that, use the jump menu to go straight to whatever is giving you trouble.

What Biotechnology Actually Means

Start with the word itself, because the word is doing all the work. Bio means living. Technology means using something to get a job done. Put them together and biotechnology is simply this: using living things, or parts of living things, to make something useful for people. That is it. No jargon required.

Notice how wide that definition is. Your grandmother setting curd overnight is using a living thing, Lactobacillus, to turn milk into something more useful. A brewer using yeast to make bread rise is doing the same. A farmer selecting the fattest cow to breed from is using a living thing to get a better outcome. All of that is biotechnology in the broad sense, and humans have been doing it for thousands of years without knowing any chemistry at all. This is what we call traditional biotechnology.

So why does the chapter feel so modern and so technical? Because somewhere in the early 1970s the game changed completely. Until then, we could only work with whatever the organism already was. We could pick a better yeast, or feed it better sugar, but we could not rewrite it. Then scientists learned to physically take a gene out of one organism and put it into another. Suddenly a bacterium could be told to make a human protein. That is modern biotechnology, and it is what this chapter is about.

The formal definitions you may meet in your textbook come from bodies like the European Federation of Biotechnology, and they are written in careful committee language. Stripped down, they say the same thing in a fussier way: biotechnology is the meeting point of pure science and engineering, in which organisms, cells, bits of cells, and even molecules built to imitate them are put to work to make products and provide services. The words products and services matter, because they remind you that biotechnology is not just laboratory curiosity. It ends in a bottle of insulin on a chemist’s shelf.

Now, an important word that will follow you through the chapter: genetic engineering. Engineering, in ordinary English, means designing and building something to a plan. Genetic engineering means designing and building a genome to a plan. You decide which gene goes where, and then you carry out the operation. The product of that operation, a DNA molecule stitched together from pieces that were never together in nature, is called recombinant DNA, and the whole set of methods used to make it is recombinant DNA technology, usually shortened to rDNA technology.

Where did all this start? The first genuinely recombinant DNA molecule was put together in 1972 by Stanley Cohen and Herbert Boyer. What they did was beautifully simple in outline: they took an antibiotic resistance gene and joined it to a plasmid, a small circular piece of DNA that lives inside bacteria. The plasmid they used came from the bacterium Salmonella typhimurium. To do it, they needed a tool that could cut DNA at a chosen place, and a tool that could glue it back. Those two tools, the cutting enzyme and the joining enzyme, are the reason this chapter exists.

Here is the everyday version. Imagine you have a long piece of ribbon printed with writing, and you want to insert one sentence from a different ribbon into the middle of it. You need scissors that cut exactly where you want, and you need tape that joins ribbon to ribbon so cleanly the join does not show. Cohen and Boyer found the biological scissors and the biological tape. Everything after 1972 is refinement.

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The Two Core Techniques Behind Modern Biotechnology

Almost every student can name the first core technique. Genetic engineering. It is the glamorous one, the one with the scissors and the plasmids. And almost every student forgets the second one, which is a shame, because examiners love it precisely because it gets forgotten.

The second core technique is this: maintaining a sterile, microbe-free environment in chemical engineering processes, so that only the organism you want grows, and it grows in enormous quantity.

Let us understand why that deserves to sit beside genetic engineering as an equal partner. Suppose you have done everything right. You have engineered a bacterium that makes human insulin. Brilliant. Now you need kilograms of insulin, not micrograms, so you put your engineered bacterium into a tank the size of a small room, full of warm nutrient broth. Warm nutrient broth is heaven for every mould spore, every stray bacterium, every yeast floating in the air of that factory. If even one contaminant gets in, it will grow alongside your engineered strain, eat your expensive nutrients, possibly outgrow your strain entirely, and pollute your product with substances you cannot easily remove.

So the whole tank, every pipe, every valve, the air going in and the air coming out, has to be sterilised and kept sterile for days on end. That is not biology, that is chemical engineering, and it is genuinely hard. This is why the syllabus calls it a core technique. Without it, genetic engineering produces nothing but an interesting experiment.

There is one more idea buried inside “genetic engineering” that is worth pulling out into daylight, because it explains why we bother with plasmids and vectors at all.

In ordinary sexual reproduction, chromosomes get shuffled. Genes from your mother and genes from your father end up combined in you. That is recombination, and it is genetic variation of a sort. But it has two crushing limitations. First, the two parents have to be of the same species, or at least close enough to interbreed. You cannot cross a human with a bacterium. Second, you have no control at all over which combination you get. It is a lottery. If you want to understand how nature’s own version of gene shuffling works, and how it differs from what we do in the lab, our notes on Principles of Inheritance and Variation lay out crossing over and independent assortment in detail.

Genetic engineering escapes both limits. It lets you move one chosen gene from any organism into any other, and it lets you choose exactly which gene. That is an enormous jump in power, and it needed a new set of tools.

Now, a problem that trips up nearly everyone. Suppose you simply inject a piece of human DNA into a bacterium. What happens to it? It sits there. When the bacterium divides, it does not get copied, because the bacterium’s copying machinery only copies DNA that carries a proper starting signal. Within a few divisions, your precious gene has been diluted away to nothing.

So the foreign DNA must become part of something that does get copied. It must either be inserted into the host’s own chromosome, or it must be attached to a small independent DNA circle that carries its own starting signal for replication. That starting signal is called the origin of replication, shortened to ori, and it is the single most important reason vectors exist. Any piece of DNA linked to an ori can be made to replicate inside a host cell. This is the idea of cloning: making many identical copies of a chosen piece of DNA.

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Restriction Enzymes: The Molecular Scissors

Picture a pair of scissors that refuses to cut anywhere you like. You hold them over a long printed ribbon and they simply will not close, until the ribbon happens to show one particular six-letter word. The moment that word appears under the blades, snip. Move along the ribbon; nothing happens; the word appears again; snip. That is a restriction enzyme. It is a molecular scissors that only cuts at one specific written word.

This fussiness is the entire point. If enzymes cut DNA anywhere, you would end up with random confetti and no way to get a whole gene out in one piece. Because a restriction enzyme cuts only at its own recognition sequence, two different scientists in two different countries can cut the same DNA and get exactly the same set of fragments. That reproducibility is what makes genetic engineering an engineering discipline rather than a lucky accident.

Now let us build the family tree properly, because questions often ask you to place restriction enzymes within it.

All enzymes that cut nucleic acids are called nucleases. Nucleases come in two flavours. Exonucleases chew nucleotides off the ends of a DNA molecule, one after another, like nibbling a biscuit from the edge inward. Endonucleases cut at points within the molecule, in the middle of the strand. Restriction enzymes are a particular kind of endonuclease, and so their full name is restriction endonuclease.

Feature	Exonuclease	Endonuclease
Where it acts	At the free ends of the DNA molecule	At internal positions within the DNA molecule
What it removes	One nucleotide at a time, progressively	Nothing is removed; the backbone is simply broken
Sequence specificity	Generally does not read a specific sequence	Restriction endonucleases read a specific recognition sequence
Use in rDNA work	Trimming ends; not used to excise genes	The workhorse: cuts out the gene and opens the vector

Where does the odd word restriction come from? It comes from a puzzle noticed in the early 1960s. Certain strains of Escherichia coli could not be infected successfully by certain bacteriophages. The bacteria were somehow restricting the growth of the virus. When this was investigated, two enzyme activities were found working together as a defence system. One enzyme added methyl groups to the bacterium’s own DNA, tagging it as “mine, do not touch”. The other enzyme cut up any DNA that lacked those methyl tags, which meant it cut up the invading viral DNA. That second, cutting enzyme was the restriction endonuclease. The first restriction endonuclease to be characterised in this way was Hind II, and it was found to cut DNA only where a particular sequence of six base pairs occurred.

So a restriction enzyme is, in its original job, a bacterial immune system. We simply borrowed it. There is something rather lovely about that: the tool at the heart of modern biotechnology was already sitting inside bacteria, doing a completely different job, for millions of years.

Since Hind II, the toolbox has become enormous. More than 900 restriction enzymes have now been isolated from over 230 strains of bacteria, and each one recognises a different sequence. Between them they give you a huge choice of places to cut.

How restriction enzymes get their names

The naming system looks cryptic until somebody decodes it once, after which it is obvious forever. Take EcoRI, the enzyme you will meet most often.

• E — the first letter of the genus, here Escherichia. It is written as a capital.
• co — the first two letters of the species, here coli. Written in small letters.
• R — the strain of the bacterium from which the enzyme was isolated. For EcoRI the strain is RY13.
• I — a Roman numeral showing the order in which that enzyme was isolated from that strain. I means it was the first one found there; a second enzyme from the same strain would be II.

The first three letters, taken from the organism’s scientific name, are conventionally written in italics because they come from a Latin binomial. The strain letter and the Roman numeral are not italicised. Examiners rarely penalise italics, but they do expect you to explain all four parts.

One practical point that students find reassuring. You do not need to memorise a list of recognition sequences. In an exam you will only ever be expected to know the EcoRI sequence, and even that is usually given to you in the question. What you must know is the logic: what the enzyme recognises, where it cuts relative to that recognition site, and what kind of ends it leaves behind. We take that up in the next section.

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Palindromic Sequences and Sticky Ends

In ordinary English, a palindrome is a word that reads the same forwards and backwards. MALAYALAM. NITIN. LEVEL. You read left to right, you read right to left, you get the same letters.

A palindrome in DNA is almost that, but with one twist that catches everybody out the first time. DNA has two strands, and the two strands run in opposite directions. One runs 5′ to 3′ left to right; its partner runs 5′ to 3′ right to left. So the rule for a DNA palindrome is:

The classic example, and the one you will be asked about, is the EcoRI site:

Check it yourself. The top strand read 5′ to 3′ is GAATTC. The bottom strand read 5′ to 3′ means starting from the right-hand end and moving left: G, A, A, T, T, C. Same word. That is a palindrome.

Now, why does nature bother making recognition sites palindromic? Here is the elegant reason. Restriction enzymes usually work as a pair of identical protein subunits, one sitting on each strand. Because the site reads identically on both strands, one enzyme design can grip both strands correctly. If the site were not palindromic, the enzyme would need two different shapes for the two strands. Symmetry in the DNA allows symmetry in the enzyme. That is the “why it works” behind palindromic recognition sites.

Where exactly does the enzyme cut?

Here is the crucial detail, and it is worth reading twice. A restriction enzyme like EcoRI does not cut through the middle of the palindrome. It cuts a little away from the centre, but it cuts between the same two bases on both strands. For EcoRI, the cut falls between G and A on each strand.

Look at what that off-centre cut produces. Each fragment now ends in a short stretch of single-stranded DNA, four bases long, hanging out with nothing paired to it: AATT on one piece, and its complement on the other. These overhanging single-stranded stretches are called sticky ends, or cohesive ends.

The name is not a metaphor invented by a textbook writer. They genuinely are sticky, in the only way DNA knows how to be sticky: by base pairing. An exposed AATT overhang will hydrogen-bond happily with any other exposed TTAA overhang it bumps into. And here is the payoff, the single most important consequence in the whole chapter:

Think of it as two pieces of a jigsaw puzzle. The enzyme is the die that stamps out the puzzle shape. Two pieces stamped by the same die will interlock. Two pieces from different puzzles will not.

Not every restriction enzyme cuts off-centre, though. Some cut straight through the middle of the palindrome, directly opposite each other on the two strands. That leaves fragments with no overhang at all, flat and fully double-stranded right up to the edge. These are blunt ends. Blunt ends can still be joined by ligase, but the reaction is far less efficient, because there is no base pairing to hold the two pieces in place while ligase does its work. There is also no specificity: any blunt end will join to any other blunt end, so unwanted combinations are common.

Point of comparison	Sticky (cohesive) ends	Blunt ends
Where the enzyme cuts	Off-centre within the palindrome, between the same two bases on both strands	Exactly at the centre of the palindrome, directly opposite on both strands
Shape of the fragment end	Short single-stranded overhang projects from each end	Fully double-stranded up to the very edge; no overhang
Base pairing before ligation	Yes — complementary overhangs hydrogen-bond and hold the pieces together	No — the two pieces must be held together by chance collision alone
Efficiency of joining	High; ligase works quickly on a pre-aligned join	Low; needs much more enzyme, more DNA and longer incubation
Specificity of joining	Only matching overhangs pair, so joins are directed	Any blunt end joins any other; unwanted products are common
Example enzyme	EcoRI, BamHI, HindIII	Hind II (cuts within its six-base-pair recognition site to leave flush ends)

Finally, meet the tape. The enzyme that seals the join is DNA ligase. Base pairing between sticky ends only holds the two fragments together with hydrogen bonds, which are weak and reversible. Ligase forms a proper covalent phosphodiester bond in the sugar-phosphate backbone, turning a temporary hold into a permanent join. Restriction enzyme and ligase are the cut-and-paste pair of molecular biology, and the finished molecule they produce is recombinant DNA.

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Cloning Vectors and What Makes a Good One

You have your gene, cut out cleanly with sticky ends on both sides. Now you have to get it into a bacterium and make sure it survives there. A bare gene will not manage that on its own. It needs a vehicle.

That vehicle is called a cloning vector, and my favourite way to describe it is this: a vector is a delivery van with its own fuel tank. It carries your cargo, the gene, into the host cell, and once inside it keeps running under its own power, copying itself again and again so that your gene gets copied with it. Without the fuel tank, the van would stop at the gate and your parcel would go nowhere.

What do we actually use as vectors? The two natural candidates that bacteria already carry are plasmids and bacteriophages. Plasmids are small circular DNA molecules that live inside a bacterium alongside the main chromosome and replicate independently of it. Bacteriophages are viruses that infect bacteria, and their DNA also replicates independently once inside. Both have the essential property that they multiply within the host cell without needing to be part of the chromosome, and some of them reach very high copy numbers — hundreds of copies per cell. If you link your gene to such a molecule, you get hundreds of copies of your gene per cell too.

Scientists then improved on nature. The plasmids used in laboratories today have been deliberately engineered so that they carry exactly the features a genetic engineer wants, and nothing that gets in the way. There are three such features, and they come up in the exam constantly.

Feature 1: Origin of replication (ori)

The ori is a specific sequence at which replication begins. It does two jobs, and students usually remember only the first.

• It starts replication. Any piece of DNA linked to an ori can be made to replicate inside the host cell. This is the fuel tank.
• It controls copy number: how many copies of the plasmid the cell ends up carrying. If you want a very large number of copies of your target DNA, you deliberately choose a vector whose ori supports a high copy number.

Feature 2: Selectable marker

Here is a problem you may not have thought about. When you mix your recombinant plasmids with a flask of bacteria, only a tiny minority of the bacteria actually take up a plasmid. Perhaps one in ten thousand. The rest are exactly as they were. So after the experiment you have a flask containing millions of ordinary bacteria and a handful of useful ones, and they all look identical.

You need a way to find the useful ones. That is the job of the selectable marker. It is a gene carried on the vector which gives the host cell some ability it did not have before, most commonly resistance to an antibiotic such as ampicillin, tetracycline, kanamycin or chloramphenicol.

Now the trick. You spread all the bacteria on a plate containing ampicillin. Any bacterium that did not take up the plasmid has no resistance gene and dies. Any bacterium that did take up the plasmid survives and grows into a visible colony. In one step you have eliminated the millions and kept the handful. Cells that have taken up foreign DNA are called transformants; those that have not are non-transformants. A selectable marker helps in identifying and eliminating non-transformants while selectively permitting the growth of transformants.

Feature 3: Cloning sites

To insert your gene, you must first open the plasmid circle with a restriction enzyme. That means the plasmid needs a recognition site for the enzyme you plan to use. But here is the catch: it needs very few such sites, and preferably only one.

Why? Because if EcoRI had three recognition sites scattered around your plasmid, cutting with EcoRI would chop the plasmid into three pieces instead of simply opening the circle. You would destroy the vector while trying to open it. So a good vector has a single, unique recognition site for each commonly used restriction enzyme, and those sites are placed deliberately at useful positions. A site of this kind is called a cloning site or recognition site.

And there is a further refinement in the positioning, which turns out to be so useful that it gets a section of its own. If the cloning site is placed inside a second marker gene, then inserting your DNA there will break that gene. That broken gene becomes a signal telling you the insertion worked. That is insertional inactivation, and we will meet it shortly.

Two further practical requirements are worth adding to your answer if the question carries good marks. A vector should be small in size, because small circles are taken up by cells far more readily and are easier to handle without breaking. And a vector should be easy to isolate from the host cell in pure form, so that you can recover the cloned DNA at the end.

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Reading the pBR322 Map

pBR322 is the textbook example of an engineered cloning vector, and once you can read this one circle, you can read any plasmid map. The name itself is a small piece of history: p stands for plasmid, BR for Bolivar and Rodriguez, the two scientists who constructed it, and 322 is simply their laboratory number for it. That is a memory aid, not an exam fact, so do not lose sleep over it.

The whole circle is about 4,361 base pairs long. Tiny, by DNA standards, which is exactly what you want in a vector. Now walk around it feature by feature.

Feature on pBR322	What it is	Why it is there
ori	Origin of replication	The sequence from which replication of the plasmid starts inside the host; it also governs how many copies of the plasmid the cell carries
ampR	Ampicillin resistance gene	A selectable marker. Cells carrying the plasmid survive on an ampicillin-containing medium; cells without it die
tetR	Tetracycline resistance gene	A second selectable marker. Because it contains the BamHI and SalI sites, it doubles as the gene that gets inactivated when foreign DNA is inserted
rop	A gene coding for proteins involved in replication of the plasmid	Works with the ori to regulate replication and therefore the copy number of the plasmid
EcoRI, ClaI, HindIII	Restriction sites lying outside both resistance genes	Cloning sites that open the plasmid without destroying either marker gene
BamHI, SalI	Restriction sites lying within tetR	Cloning here inactivates tetracycline resistance, which is used to identify recombinants
PstI, PvuI	Restriction sites lying within ampR	Cloning here inactivates ampicillin resistance instead; selection then uses tetracycline

Notice the design cleverness here. The plasmid carries two different antibiotic resistance genes, and each one contains its own restriction sites. That gives the experimenter a choice. Insert into tetR using BamHI, and tetracycline resistance breaks while ampicillin resistance stays intact. Insert into ampR using PstI, and it is the other way round. Either way you always have one working marker to select transformants with, and one broken marker to identify recombinants with. That is not an accident; it is the whole point of the design.

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Insertional Inactivation and Blue-White Selection

We now have to solve a problem that is one level trickier than the one the selectable marker solved. Read this slowly, because getting the two problems clearly separated is what makes this section easy.

Problem one was: which cells took up a plasmid at all? Solved by the antibiotic marker. Problem two is: among the cells that took up a plasmid, which ones took up a plasmid that actually contains my gene?

Why is that a real problem? Because when you cut a plasmid open with a restriction enzyme and then add ligase, not every plasmid picks up an insert. Many of them simply close back up on themselves, exactly as they were before. These are called non-recombinants, and they carry a perfectly functional antibiotic resistance gene, so they grow on your selection plate just as happily as the ones you want. The antibiotic cannot tell them apart.

The solution is beautifully simple: insertional inactivation. Put the cloning site inside a gene whose product you can detect. If foreign DNA gets inserted there, the gene is broken in half and stops working. If nothing gets inserted, the gene works normally. Now the state of that gene is a report on whether insertion happened.

Think of it as a security seal on a jar. An unbroken seal means nobody opened it. A broken seal means somebody did. In insertional inactivation, we deliberately arrange for the seal to break whenever our gene goes in.

Version 1: inactivating an antibiotic resistance gene

Take pBR322 and insert foreign DNA at the BamHI site, which sits inside tetR. Now:

• Untransformed cells (no plasmid at all) — die on ampicillin.
• Non-recombinants (plasmid re-closed, no insert) — grow on ampicillin and grow on tetracycline.
• Recombinants (plasmid carries the insert) — grow on ampicillin but do not grow on tetracycline, because tetR is broken.

So you plate the colonies on ampicillin, then transfer copies of the same colonies in the same pattern onto tetracycline, and look for colonies that are present on the first plate but missing from the second. Those are your recombinants.

It works, and it is examinable. But be honest about its drawback: it is cumbersome. You need two plates, you need to transfer the colonies faithfully so the patterns match up, and you identify the cells you want by the fact that they are absent from the second plate — which means you then have to go back to the first plate to actually collect them. Fiddly, slow, and error-prone.

Version 2: blue-white selection

Here is the elegant improvement. Instead of putting the cloning site inside an antibiotic resistance gene, put it inside a gene coding for the enzyme β-galactosidase, whose gene is called lacZ.

β-galactosidase can break down a specially made substrate called a chromogenic substrate. The word chromogenic means colour-producing: when the enzyme cuts this substrate, one of the products is a bright blue compound. So if you add the chromogenic substrate to the growth medium:

• Non-recombinant colonies — lacZ is intact, β-galactosidase is made, the substrate is broken down, and the colony turns blue.
• Recombinant colonies — foreign DNA sits in the middle of lacZ, so no functional β-galactosidase is made, the substrate is untouched, no blue compound forms, and the colony stays white.

One plate. One glance. The white colonies are the ones you want. No replica plating, no matching up patterns, no working out which colony is missing. You simply pick the white ones with a loop and carry on.

Point of comparison	Antibiotic-based insertional inactivation	Blue-white selection
Gene inactivated	An antibiotic resistance gene, for example tetR	lacZ, the gene for β-galactosidase
What is added to the medium	The antibiotic itself	A chromogenic substrate
How recombinants appear	They fail to grow on the second antibiotic plate	They grow as white colonies on the same plate
Number of plates needed	Two, requiring simultaneous plating and faithful transfer	One
Practical verdict	Works, but cumbersome and easy to get wrong	Faster, simpler and now the preferred method

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Vectors for Plants and Animals

Plasmids are wonderful for bacteria. But suppose you want to put a gene into a tomato plant, or into a human cell. A plasmid on its own will not get in and will not stay. You need a delivery system that already knows how to break into that kind of cell. And here is the recurring idea of this section, which is genuinely one of the most satisfying in the chapter:

For plants: the Ti plasmid of Agrobacterium tumefaciens

Agrobacterium tumefaciens is a soil bacterium that infects many dicot plants and causes a disease called crown gall, in which a swollen tumour-like growth appears at the base of the stem. The bacterium does this by transferring a piece of its own DNA into the plant cell, where it integrates into the plant’s genome and forces the plant cell to divide uncontrollably.

That transferred DNA is carried on a plasmid called the Ti plasmid. Ti stands for tumour inducing. So Agrobacterium is, in effect, nature’s own genetic engineer — it has been transferring genes into plant cells for millions of years.

What scientists did was to modify the Ti plasmid: remove the tumour-causing genes and put the gene of interest in their place. The modified plasmid can no longer cause crown gall disease, but it retains its ability to deliver DNA into a plant cell and get that DNA integrated into the plant genome. The Ti plasmid has thus been converted from a pathogen’s weapon into a cloning vector suitable for delivering desired genes into plants.

For animals: disarmed retroviruses

Retroviruses are a group of viruses that infect animal cells, and in some cases their infection transforms a normal cell into a cancerous one. Like Agrobacterium, they achieve this by inserting their genetic material into the host cell’s genome, where it becomes a permanent part of the cell’s DNA.

Once again, the trick is to disarm them. Remove the genes responsible for causing cancer, insert the desired gene, and you are left with a vector that is very good at delivering that gene into animal cells and getting it stably integrated, without turning the cell cancerous. If you would like to see what happens when this is used in medicine, our notes on Human Health and Disease cover the immunological background that gene therapy and modern vaccines rest on.

Vector	Natural source and natural role	Target host	How it is modified for use
Plasmid (e.g. pBR322)	Small circular DNA found naturally in bacteria; replicates independently of the chromosome	Bacteria such as E. coli	Engineered to carry an ori, selectable markers and single cloning sites
Bacteriophage	A virus that infects bacteria; its DNA multiplies to a high copy number inside the cell	Bacteria	Non-essential viral genes replaced by the DNA to be cloned; useful for larger inserts
Ti plasmid	Tumour-inducing plasmid of Agrobacterium tumefaciens; causes crown gall in dicot plants	Plant cells	Tumour-causing genes removed and replaced with the gene of interest; the plasmid is thereby disarmed
Retrovirus	Animal virus that integrates its genetic material into the host genome; some transform normal cells into cancerous cells	Animal cells	Cancer-causing genes removed and the desired gene inserted; used to deliver genes stably into animal cells

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Making a Host Cell Competent

You have a recombinant plasmid in a tube. You have a flask of E. coli. Mix them and… nothing happens. The bacteria carry on as before and the plasmid stays in solution.

Why? Because DNA is a large, heavily negatively charged molecule, thanks to all those phosphate groups in its backbone. The bacterial cell wall and membrane are themselves negatively charged, and like charges repel. Beyond that, a bacterium’s outer layers are simply not designed to let big hydrophilic molecules wander in. So under ordinary conditions a bacterium cannot take up DNA from its surroundings.

To make it possible, the host cell has to be made competent — that is, temporarily made able to take up DNA. Competence is not a permanent property; it is a state we force the cell into.

The calcium-and-heat-shock method

This is the standard laboratory method for bacteria, and it comes in three moves.

1. Treat with a divalent cation. The cells are suspended in a solution containing a specific concentration of a divalent cation, most commonly calcium (Ca²⁺). This increases the efficiency with which DNA passes through the pores of the bacterial cell wall. Chemically, the positively charged calcium ions sit between the negatively charged DNA and the negatively charged cell surface, neutralising the repulsion and letting the DNA come close.
2. Incubate on ice with the recombinant DNA. The cells and the plasmid are held together at low temperature so that the DNA gathers at the cell surface.
3. Give a brief heat shock, then return to ice. The mixture is warmed suddenly to about 42°C for a few seconds and then put straight back on ice. The abrupt temperature change disturbs the membrane just enough for the DNA to slip through. Returning to ice closes things up again and lets the cell recover.

Think of the cold-hot-cold sequence as briefly startling the cell. A door that is normally shut is jolted open for an instant, and the plasmid takes its chance.

Other ways of getting DNA into a cell

Calcium and heat shock work well for bacteria, but plant and animal cells need other approaches.

• Microinjection. Recombinant DNA is injected directly into the nucleus of an animal cell using an extremely fine glass needle under a microscope. Precise, but one cell at a time.
• Biolistics, also called the gene gun. Cells, usually plant cells, are bombarded with high-velocity microparticles of gold or tungsten that have been coated with the DNA. The particles punch through the cell wall carrying the DNA with them. It is exactly what it sounds like: shooting DNA into cells.
• Disarmed pathogen vectors. As described above, the disarmed Ti plasmid and disarmed retroviruses deliver the DNA themselves. Here you are not forcing the DNA in; you are letting a professional do the job.

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Tools done. You now know about the scissors, the delivery van and the doorway. From here on, the chapter is simply a walk through the seven steps of the process, in order. Everything you meet from now on uses only the tools you have already learned.

Step 1: Isolating the Genetic Material

Before you can cut a gene out, you have to have the DNA in your hand, in a tube, free of everything else the cell contained. That is harder than it sounds, because DNA inside a living cell is not floating about loose. It is wrapped in proteins, mixed with RNA, surrounded by lipids and sugars, and locked behind a cell wall and a membrane. Step 1 is the demolition and clean-up job.

Work through it in the order the obstacles appear.

(a) Break open the cell

Each type of cell has a different outer covering, so each needs a different enzyme to break it down. This is a favourite one-mark question, so learn the pairings exactly.

Cell type	Main component of the wall	Enzyme used to break it
Bacterial cell	Peptidoglycan	Lysozyme
Plant cell	Cellulose	Cellulase
Fungal cell	Chitin	Chitinase

Notice how the enzyme names simply take the name of the material and add -ase. Cellulose to cellulase. Chitin to chitinase. Once you spot that pattern, there is nothing left to memorise except lysozyme, which is the odd one out because it is named after what it does, not what it eats.

(b) Get rid of everything that is not DNA

Once the cell is broken open, its whole contents spill out as a soup. In that soup, along with your DNA, are RNA molecules, proteins, polysaccharides and lipids. Each is removed with a treatment aimed at it.

• RNA is removed by treatment with ribonuclease (RNase). This is essential: RNA is chemically very like DNA, so it would contaminate every later step and confuse every measurement.
• Proteins are removed by treatment with protease. Remember that in a eukaryotic cell the DNA is wound tightly around histone proteins; until the proteins are digested away, the DNA is not really free.
• Other molecules such as polysaccharides and lipids are removed by suitable further treatments.

If you want to remind yourself exactly how DNA is packaged with histones into nucleosomes and chromatin, and why that packaging matters here, our Molecular Basis of Inheritance notes cover it step by step.

(c) Precipitate the purified DNA

Now the satisfying bit. Add chilled ethanol to the cleaned-up solution. DNA does not dissolve in ethanol, so it comes out of solution and can be seen with the naked eye as a mass of fine white threads suspended in the liquid, rather like very thin cotton wool.

Why chilled? Cold ethanol makes the precipitation more complete and helps the DNA come out as long intact threads rather than a fine haze. And the reason ethanol works at all is worth understanding: water molecules normally surround the charged phosphate backbone and keep DNA dissolved. Ethanol pushes that water away, the DNA molecules clump together, and out they come.

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Step 2: Cutting the DNA at Specific Locations

You have pure DNA in a tube. Now the scissors come out. This step is where all that theory about restriction enzymes and sticky ends becomes an actual laboratory procedure.

The procedure itself is unglamorous. Restriction enzyme digestion is carried out by incubating the purified DNA with the chosen restriction enzyme, at the optimum conditions for that enzyme — the right buffer, the right salt concentration, and typically a temperature near 37°C, which is where most of these bacterial enzymes work best. You put it in a water bath and wait.

What matters is the logic of what you cut and with what.

1. Cut the source DNA — the DNA containing your gene of interest — with your chosen restriction enzyme. This releases the gene as a fragment with sticky ends.
2. Cut the vector DNA with the same enzyme. This opens the plasmid circle at its single cloning site, producing a linear molecule with the same sticky ends.
3. Mix the two. Complementary overhangs find each other and pair by hydrogen bonding.
4. Add DNA ligase. Ligase seals the nicks in the sugar-phosphate backbone with covalent phosphodiester bonds. The result is a closed circular molecule made of vector DNA plus your insert — a recombinant DNA molecule.

How do you know the digestion actually worked? You cannot see DNA in a tube. So the progress of a restriction enzyme digestion is checked using agarose gel electrophoresis, which is the whole of Step 3. Cutting and checking are inseparable in practice, which is why the two steps are usually taught together.

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Step 3: Separating and Checking Fragments by Gel Electrophoresis

After digestion your tube contains a mixture of DNA fragments of different lengths. You need to know whether the cut worked, and you need to pull out the one fragment you actually want. Both jobs are done by the same technique: agarose gel electrophoresis.

The name breaks into two halves that explain themselves. Electro means electricity. Phoresis means being carried along. So electrophoresis is separating things by making them travel in an electric field. Agarose simply tells you what the track is made of: agarose is a natural polymer extracted from seaweed, which sets into a firm jelly full of microscopic pores.

Two facts make the whole thing work.

• DNA is negatively charged. Every nucleotide carries a phosphate group with a negative charge. So in an electric field, every DNA fragment moves towards the positive electrode, the anode. No exceptions, no matter what the sequence is.
• The gel is a sieve. Because the agarose jelly is full of tiny pores, a fragment must thread its way through. Short fragments squeeze through easily and travel far. Long fragments get caught up and travel only a short distance in the same time.

Put those together and you have the central rule: the smaller the fragment, the further it moves. After running the gel for a while, fragments of the same size have all travelled the same distance and have collected together in a narrow line across the gel. Each line is called a band.

A helpful mental picture: imagine a crowd of people of different sizes trying to run through a dense forest. The small, agile ones get furthest. The large ones get tangled in the branches. After ten minutes, the crowd has sorted itself by size without anyone measuring anybody.

Seeing the invisible

DNA is colourless, so the separated bands are invisible in an ordinary gel. To see them, the gel is stained with ethidium bromide and then exposed to ultraviolet radiation. Ethidium bromide slips in between the base pairs of the DNA and, under UV light, fluoresces. The bands then appear as bright orange bands sitting in a dark gel. Without the stain and the UV, you would see nothing at all.

Elution

Now you can see your fragment, but it is still stuck inside a slab of jelly. The last move is to cut that band out of the gel with a blade and extract the DNA from the gel piece. This extraction of the separated DNA fragment from the gel is called elution. The DNA fragments recovered by elution are pure and ready to be used in the next stage — joining to the vector to make recombinant DNA.

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Step 4: Amplifying the Gene of Interest Using PCR

PCR stands for Polymerase Chain Reaction, and it is probably the single most useful technique ever invented in molecular biology. Here is the problem it solves.

You have isolated your gene, but you have almost none of it — perhaps a few molecules. You cannot do anything with a few molecules. You need millions. PCR is a photocopier that copies only the page you bookmarked. You do not have to photocopy the whole book; you place two bookmarks on the page you want, and the machine copies only what lies between them, over and over again.

Formally: PCR is the synthesis of multiple copies of a gene, or a piece of DNA, of interest in vitro, using two sets of primers and the enzyme DNA polymerase.

The bookmarks are called primers. A primer is a short chemically synthesised stretch of nucleotides, usually around twenty bases long, whose sequence is complementary to the sequence at one end of the region you want copied. You need two: one for each strand, one marking each end. Because DNA polymerase can only add nucleotides to an existing chain and cannot start one from nothing, the primer provides the free end from which building begins. That is why they are called primers — they prime the reaction.

The three steps of one cycle

Step	Typical temperature	What physically happens	Why that temperature
1. Denaturation	About 94°C	The hydrogen bonds between the two strands break, and the double-stranded DNA separates into two single strands, each of which will act as a template	High heat is needed to overcome the many hydrogen bonds holding the helix together; the covalent backbone is unaffected
2. Annealing	About 50°C to 55°C	The two primers base-pair with their complementary sequences at either end of the target region, one on each template strand	Cool enough for short primers to bind, but still warm enough that the two long template strands do not simply re-join with each other
3. Extension	About 72°C	DNA polymerase extends each primer using the four deoxynucleotides supplied in the reaction, synthesising the region between the primers	This is the temperature at which Taq polymerase works fastest and most accurately

At the end of one cycle, every double-stranded molecule has become two. Run the cycle again and two become four, then eight, then sixteen. This doubling is why it is called a chain reaction. Repeat it about thirty times and the arithmetic becomes astonishing: the gene of interest is amplified to approximately one billion copies.

Why Taq polymerase, and why it matters

This is the detail that turns PCR from an idea into a practical machine, and it is a favourite exam question.

Every cycle begins by heating the mixture to about 94°C. An ordinary DNA polymerase — the sort found in you, or in E. coli — is a protein, and proteins denature at that temperature. It would be destroyed in the first cycle, and you would have to open the tube and add fresh enzyme before every single round. That would make PCR unusable.

The solution came from a hot spring. Thermus aquaticus is a bacterium that lives in extremely hot water, and its DNA polymerase, called Taq polymerase, is thermostable: it remains active despite the high temperature used to denature the double-stranded DNA. Add it once at the start, and it survives every cycle. That single property is what allows the whole reaction to be automated in a machine.

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Step 5: Inserting Recombinant DNA into the Host

Everything up to now has happened in a tube. The recombinant DNA molecule exists, but it is inert. To make it do anything, it has to get inside a living cell that will copy it and read it.

You already know the mechanics of this from the section on competent cells. The recombinant DNA is mixed with host cells that have been made competent by treatment with a divalent cation such as calcium, and the mixture is taken through the ice, brief heat shock at about 42°C, and back to ice sequence. Some of the cells take up the recombinant plasmid. Those cells are now transformed, and the process is called transformation: a procedure through which a piece of DNA is introduced into a host bacterium.

What follows immediately afterwards is the selection you met earlier, and it is worth restating here because in an exam it belongs to this step.

1. Spread the treated cells on a medium containing the antibiotic whose resistance gene the vector carries. Only transformants survive; untransformed cells die.
2. Among the survivors, distinguish recombinants from non-recombinants by insertional inactivation — either by the loss of a second antibiotic resistance, or, more conveniently, by picking the white colonies on a plate containing a chromogenic substrate.
3. Pick a single recombinant colony and grow it up. Because every cell in a colony has descended from one original cell, every cell in it carries the same recombinant plasmid. You now have a clone.

There is a lovely worked illustration of this in the classic pBR322 experiment. Ligate foreign DNA at the BamHI site of tetR. The resulting recombinant plasmid loses tetracycline resistance but keeps ampicillin resistance. Plate on ampicillin: everything that took up a plasmid grows. Transfer to tetracycline: the recombinants alone fail to grow. The colonies that grow on ampicillin but not on tetracycline are exactly the ones you want, and you go back to the ampicillin plate to collect them.

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Step 6: Obtaining the Foreign Gene Product and Bioreactors

Your recombinant bacterium is now happily multiplying in a small flask, making the protein you engineered it to make. Wonderful — except that a flask gives you milligrams and the world needs kilograms. Step 6 is the jump from laboratory to factory.

First, a distinction that examiners like. If the aim is simply to produce the protein, you do not necessarily have to grow the cells yourself; the gene can be expressed in a heterologous host and the protein harvested. But in almost all practical manufacturing, cells are grown in large volumes. A small flask, shaken continuously so that air keeps mixing in, will do for a few hundred millilitres. Beyond that, you need a bioreactor.

A bioreactor is a vessel in which raw materials are biologically converted into specific products, using microbial, plant, animal or human cells. Bioreactors handle large volumes — typically 100 to 1000 litres and often much more — and they provide the optimum conditions for a cell to grow: temperature, pH, substrate, salts, vitamins and oxygen.

The most commonly used bioreactor is the stirred-tank type. It is usually cylindrical, or has a curved base, so that the contents mix thoroughly rather than settling in corners. A stirrer sits inside, and it does two jobs at once: it mixes the contents evenly, and it keeps oxygen available to every cell throughout the vessel.

Why does oxygen deserve special attention? Because oxygen dissolves poorly in water, and a dense culture of bacteria consumes it faster than it can diffuse in. Cells starved of oxygen stop producing. So aeration is a constant engineering challenge, and it is what separates the two types of stirred-tank bioreactor you are asked to compare.

Point of comparison	Simple stirred-tank bioreactor	Sparged stirred-tank bioreactor
How air is supplied	Oxygen enters at the surface and is drawn into the liquid by the action of the stirrer alone	Air is bubbled through the culture from below, through a device called a sparger
Role of the stirrer	Mixes the contents evenly and facilitates the availability of oxygen throughout the vessel	Mixes the contents and also breaks the rising bubbles into smaller ones, increasing the surface for gas exchange
Oxygen transfer rate	Lower; adequate for less demanding cultures	Higher; suits dense, fast-growing, highly aerobic cultures
Distinguishing part	No sparger present	A sparger at the base releases fine air bubbles

Whichever type is used, a bioreactor carries the same set of standard fittings, and these make a very common short-answer question.

• An agitator system — the motor, shaft and impellers that mix the contents.
• An oxygen delivery system — supplies sterile air or oxygen to the culture.
• A foam control system — vigorous stirring and aeration of a protein-rich broth produces foam, which reduces the working volume and can block filters; antifoam agents and mechanical breakers control it.
• A temperature control system — enzymes work within a narrow temperature range, and fermentation itself generates heat that must be removed.
• A pH control system — metabolism releases acids and bases that would otherwise drive the pH out of the optimal range.
• Sampling ports — allow small volumes of culture to be withdrawn periodically, without breaking sterility, so that growth and product formation can be monitored.

If you would like to see how these same vessels are used for products that have nothing to do with genetic engineering — antibiotics, enzymes, organic acids, fermented beverages — our notes on Microbes in Human Welfare cover industrial microbial products in detail, and the two chapters reinforce each other nicely.

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Step 7: Downstream Processing

The bioreactor has finished its run. Inside is a warm, cloudy soup of billions of cells, spent nutrients, waste products — and, somewhere in there, the protein you have spent the whole chapter trying to make. Getting it out, and getting it clean enough to inject into a human being, is downstream processing.

The word simply means everything that happens after the biosynthesis is complete, before the product is ready to be marketed. It has four parts.

1. Separation. The product is separated from the cells, the culture medium and everything else in the vessel. If the protein has been secreted, the cells are removed and the liquid kept; if it stays inside the cells, the cells are collected and broken open.
2. Purification. The separated material still contains many other cellular proteins and molecules. These are removed until only the desired product remains.
3. Formulation with preservatives. The purified product is formulated with suitable preservatives so that it remains stable and does not become contaminated between manufacture and use.
4. Clinical trials and quality control. If the product is a drug, it has to undergo thorough clinical trials before it can be given to patients. Every batch also passes through strict quality control testing.

Two things about downstream processing are worth remembering because they are frequently examined as a single line of reasoning.

And the second point: this stage often costs more than everything before it. Making the protein is, in a sense, the easy part. Cleaning it up to injectable purity, proving it is safe, and demonstrating batch after batch that it meets specification is where most of the money and most of the years go. Students often skim this section because it sounds like paperwork. Examiners include it precisely because it is the step that turns a laboratory result into a medicine somebody can actually buy.

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Putting the Whole Workflow Together

Read the flowchart from left to right along the top row, drop down at Step 4, then read right to left along the bottom row. Seven notes, one story. If you can reproduce those seven boxes from memory, with one sentence each, you can answer almost any long question in this chapter.

Here is the whole thing as a single narrative, which is what you should be able to write in a five-mark answer.

The genetic material is isolated by breaking the cell open with the appropriate enzyme and digesting away RNA and protein, and the DNA is then precipitated with chilled ethanol. The gene of interest and the vector are both cut with the same restriction enzyme, so that both carry identical sticky ends. The digestion is checked and the required fragment recovered by agarose gel electrophoresis followed by elution. The gene is amplified to about a billion copies by PCR, using two primers and thermostable Taq polymerase across roughly thirty cycles of denaturation, annealing and extension. The amplified fragment is ligated into the vector by DNA ligase, and the recombinant DNA is introduced into a competent host cell, usually by calcium treatment and a brief heat shock. Transformants are selected using the antibiotic marker and recombinants identified by insertional inactivation. The selected clone is then grown on a large scale in a bioreactor supplying controlled temperature, pH, oxygen and nutrients, so that the foreign gene product accumulates. Finally, the product is separated, purified, formulated with preservatives and subjected to clinical trials and quality control before it is marketed.

Notice how many of the tools appear more than once. The restriction enzyme is used twice, on the gene and on the vector. Gel electrophoresis is used both to check the cut and to purify the fragment. The selectable marker is used at Step 5 but was designed into the vector back at the very beginning. Good design at the start makes the later steps easy — which is, when you think about it, the definition of engineering. And if you are curious how these same tools reshaped our understanding of how species change over time, the story of molecular evidence is picked up in our Evolution chapter notes.

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Biotechnology Principles and Processes Class 12 Important Questions — Practice Worksheet

Ten questions, all original, arranged roughly in increasing difficulty. Do them closed-book with a blank sheet and a pen. Only then open the answers. Reading a model answer feels productive and teaches you almost nothing; writing your own answer first and then comparing is where the learning actually happens.

1. A restriction enzyme is isolated from a bacterium and named BsuII. Expand each part of this name. (2 marks)

Show Answer

The capital B is the first letter of the genus, in this case Bacillus. The small letters su are the first two letters of the species name, here subtilis. Together, the first three letters identify the organism from which the enzyme was isolated, and are conventionally written in italics. (1 mark.)

The Roman numeral II indicates the order in which the enzyme was isolated from that particular strain of the bacterium — here, the second enzyme isolated from it, in sequence of discovery. Where a strain letter is present in such a name, it identifies the strain; in this name no separate strain letter is given. (1 mark.)

2. Differentiate between sticky ends and blunt ends, and state which is preferred for cloning and why. (3 marks)

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Sticky ends are produced when a restriction enzyme cuts the two strands at points away from the centre of the palindrome, though between the same two bases on each strand. Each fragment is left with a short single-stranded overhang. (1 mark.)

Blunt ends are produced when the enzyme cuts both strands exactly opposite each other, at the centre of the recognition sequence, so the fragment is fully double-stranded right to its edge with no overhang. (1 mark.)

Sticky ends are preferred for cloning. Their overhangs are complementary, so a cut vector and a cut insert produced by the same enzyme base-pair with each other spontaneously and are held in alignment while DNA ligase seals the backbone. This makes ligation both far more efficient and far more specific than blunt-end joining, in which any end can join any other end. (1 mark.)

3. Why must the vector and the source DNA be cut with the same restriction enzyme? (2 marks)

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A given restriction enzyme recognises one specific palindromic sequence and always cuts it at the same position, so every fragment it produces carries the same single-stranded overhang. Cutting both the vector and the source DNA with that same enzyme therefore gives both molecules identical, mutually complementary sticky ends. (1 mark.)

When the two are mixed, these complementary overhangs base-pair with each other by hydrogen bonding and hold the pieces in the correct alignment, allowing DNA ligase to seal the sugar-phosphate backbone and produce a stable recombinant molecule. If different enzymes were used, the overhangs would not be complementary, no pairing would occur and ligation would essentially fail. (1 mark.)

4. Explain the role of the origin of replication in a cloning vector, and state one consequence of choosing a vector with a high copy number ori. (3 marks)

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The origin of replication, or ori, is the specific sequence at which replication of a DNA molecule begins inside the host cell. Any piece of DNA that is linked to an ori can be made to replicate within that host. (1 mark.)

Without an ori, foreign DNA introduced into a bacterium is not copied when the cell divides, so it is progressively diluted out and lost. The ori is therefore the feature that turns a passive piece of DNA into something that multiplies. (1 mark.)

The ori also controls the copy number, that is, how many copies of the vector each cell maintains. Choosing a vector with a high copy number ori means each cell carries many copies of the recombinant plasmid, so a very large number of copies of the target DNA, and a correspondingly large amount of its protein product, can be obtained from a given culture. (1 mark.)

5. Distinguish between a transformant and a recombinant, and explain how each is identified. (3 marks)

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A transformant is a host cell that has taken up foreign DNA of any kind, including a vector that carries no insert. A recombinant is a cell carrying a vector into which the desired foreign DNA has actually been ligated. Every recombinant is a transformant, but not every transformant is a recombinant. (1 mark.)

Transformants are identified using the selectable marker on the vector. The cells are grown on a medium containing the corresponding antibiotic; untransformed cells lack the resistance gene and die, while all transformants survive and form colonies. (1 mark.)

Recombinants are identified by insertional inactivation. Either a second antibiotic resistance gene containing the cloning site is disrupted by the insert, so recombinants fail to grow on that second antibiotic, or the lacZ gene is disrupted, so recombinant colonies remain white on a medium containing a chromogenic substrate while non-recombinants turn blue. (1 mark.)

6. A student inserts foreign DNA into the PstI site of pBR322. Which resistance is lost, which is retained, and how would you now select recombinants? (3 marks)

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The PstI site lies within the ampicillin resistance gene, ampR. Inserting foreign DNA there interrupts that gene, so ampicillin resistance is lost. The tetracycline resistance gene, tetR, is untouched and remains functional, so tetracycline resistance is retained. (1 mark.)

Selection must therefore be done the other way round from the usual BamHI example. The transformed cells are first plated on a medium containing tetracycline. All cells that have taken up any form of the plasmid grow; untransformed cells die. (1 mark.)

The surviving colonies are then transferred, in the same pattern, onto a medium containing ampicillin. Colonies that grow on tetracycline but fail to grow on ampicillin are the recombinants, since their ampR gene has been inactivated by the insert. The corresponding colonies are then picked from the original tetracycline plate. (1 mark.)

7. Why is a heat shock at about 42°C given during bacterial transformation, and why are the cells kept on ice before and after it? (3 marks)

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DNA is a large, negatively charged, hydrophilic molecule and cannot cross the bacterial cell envelope under normal conditions, so the cells must first be made competent. They are treated with a specific concentration of a divalent cation, usually calcium, which neutralises the repulsion between the negatively charged DNA and the negatively charged cell surface and increases the efficiency with which DNA passes through pores in the cell wall. (1 mark.)

The cells and recombinant DNA are held together on ice so that the DNA accumulates at the cell surface while the membrane stays relatively rigid and stable. (1 mark.)

The brief heat shock at about 42°C creates a sudden change in the membrane that makes it transiently permeable, allowing the recombinant DNA to enter. Returning the cells immediately to ice ends that permeable state and allows them to recover, so that they survive and can go on to divide. (1 mark.)

8. Describe the three steps of one PCR cycle, giving the approximate temperature and the purpose of each. (3 marks)

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Denaturation, at about 94°C. The hydrogen bonds holding the two strands of the double helix together are broken and the DNA separates into two single strands. The covalent backbone of each strand is unaffected, so each separated strand can act as a template. (1 mark.)

Annealing, at about 50°C to 55°C. The mixture is cooled so that two chemically synthesised primers, one complementary to each template strand, base-pair with the sequences flanking the region to be copied. The primers define the exact stretch that will be amplified and provide the free ends from which DNA polymerase can begin. (1 mark.)

Extension, at about 72°C. Thermostable Taq polymerase, obtained from Thermus aquaticus, extends each primer using the supplied deoxynucleotides and synthesises the complementary strand. At the end of the cycle each original molecule has become two, and repeating the cycle about thirty times amplifies the gene roughly a billion times. (1 mark.)

9. Compare a simple stirred-tank bioreactor with a sparged stirred-tank bioreactor, and explain why aeration matters so much. (3 marks)

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In a simple stirred-tank bioreactor, the vessel is usually cylindrical or has a curved base to help mixing, and a stirrer is used to mix the contents evenly and to facilitate the availability of oxygen throughout the vessel. Oxygen enters mainly at the liquid surface and is drawn in by the stirring action. (1 mark.)

In a sparged stirred-tank bioreactor, air is additionally bubbled through the culture from a sparger at the base of the vessel. The stirrer both mixes the broth and breaks the rising bubbles into smaller ones, greatly increasing the surface available for gas exchange and therefore the rate at which oxygen dissolves. (1 mark.)

Aeration matters because oxygen is only sparingly soluble in water, while a dense culture of aerobic cells consumes it very rapidly. If oxygen becomes limiting, growth slows and synthesis of the desired product falls, so the oxygen delivery system is one of the essential control systems of any bioreactor, alongside agitation, foam control, temperature control, pH control and sampling ports. (1 mark.)

10. Arrange the seven steps of recombinant DNA technology in the correct order and write one sentence on each. (5 marks)

Show Answer

(i) Isolation of the genetic material. The cell is broken open using lysozyme, cellulase or chitinase as appropriate; RNA is removed with ribonuclease and protein with protease; and the purified DNA is precipitated as fine threads by adding chilled ethanol. (1 mark.)

(ii) Cutting the DNA at specific locations. The source DNA and the vector are both digested with the same restriction endonuclease, so that both acquire identical complementary sticky ends. (1 mark.)

(iii) Separation and isolation of DNA fragments. The digest is run on an agarose gel, where the negatively charged fragments migrate towards the anode and are separated by size; the required band is stained, located under ultraviolet light and recovered from the gel by elution.

(iv) Amplification using PCR. The gene is copied about a billion times through roughly thirty cycles of denaturation, annealing and extension, using two primers and thermostable Taq polymerase. (1 mark for (iii) and (iv) together.)

(v) Insertion of recombinant DNA into the host. The amplified fragment is ligated into the vector with DNA ligase, and the recombinant DNA is introduced into competent host cells by calcium treatment followed by a brief heat shock; transformants and then recombinants are selected. (1 mark.)

(vi) Obtaining the foreign gene product. The selected recombinant clone is cultured on a large scale in a bioreactor that supplies optimum temperature, pH, oxygen, substrate, salts and vitamins, so that the foreign gene is expressed and its product accumulates.

(vii) Downstream processing. The product is separated from the cells and medium, purified, formulated with suitable preservatives, and subjected to clinical trials where applicable and to strict quality control before it is marketed. (1 mark for (vi) and (vii) together.)

Note on marking: the order itself carries a mark in most schemes. Even if your detail is thin, get the sequence right.

Mark yourself honestly. Anything you scored below full marks on goes on a small list, and that list — not the whole chapter — is what you revise tomorrow.