Chapter 9: Biotechnology: Principles and Processes (Biotechnology)

For example, insulin (diabetes), growth hormone, interferons, erythropoietin, tPA (clots), factor VIII (haemophilia), hepatitis B vaccine, interleukin-2 and ADA.

Restriction enzyme: EcoRI (from Escherichia coli RY 13).

Substrate DNA and recognition site (a palindrome):

5′-G AATTC-3′ 3′-CTTAA G-5′

Cut: EcoRI cuts each strand between G and A, at the same position on both strands (reading 5′→3′).

Product: two DNA fragments with single-stranded overhanging ends, called sticky ends:

5′-G and AATTC-3′ on one fragment pair, with complementary 3′-CTTAA and G-5′ on the other.

These sticky ends can pair with any other DNA cut by EcoRI and be joined by DNA ligase, forming recombinant DNA.

EcoRI recognises 5′-GAATTC-3′, cuts between G and A on both strands, and gives fragments with AATT sticky ends.

DNA is bigger. A DNA molecule is made of millions (in humans about 3 × 10⁹ base pairs per haploid set) of nucleotides and can be centimetres long when stretched; it can be seen as threads when precipitated with chilled ethanol. An enzyme is a protein of a few hundred to a few thousand amino acids. Also, enzymes like restriction enzymes move along and cut DNA at specific sites, showing DNA is much larger.

DNA is far bigger; it has millions of nucleotides (visible as threads with ethanol), while enzymes have only hundreds of amino acids.

A diploid human cell has about 6.6 × 10⁹ base pairs of DNA. In moles:

$$\frac{6.6\times 10^9}{6.022\times 10^{23}}\approx 1.1\times 10^{-14}\text{mol of base pairs}$$

The nucleus has a volume of roughly $5\times 10^{-13}$ L, so the concentration is about

$$\frac{1.1\times 10^{-14}}{5\times 10^{-13}}\approx 0.02\text{M (as base pairs)}$$

(If we count the 46 DNA molecules instead, it is $\frac{46}{6.022\times 10^{23}}$ mol, a concentration of only about $1.5\times 10^{-10}$ M.) The exact value depends on the cell volume taken.

About 1.1 × 10⁻¹⁴ mol of base pairs per cell, roughly 0.02 M (as base pairs) in the nucleus.

No (in general). Restriction endonucleases are found in bacteria, where they defend against invading viral (phage) DNA by cutting it; the bacterium protects its own DNA by methylation. Eukaryotic cells do not have such restriction–modification systems; their DNA is protected inside the nucleus and they have other defence mechanisms.

No; restriction enzymes are a bacterial defence against phage DNA.

• They handle large volumes (100–1000 litres or more) for industrial production.
• They have systems to control temperature, pH, oxygen and foam, and sampling ports to monitor the culture.
• Nutrients can be added and products removed under sterile conditions (continuous culture).
• So growth conditions stay optimal and yields are higher.

Large scale, and control of temperature, pH, oxygen, foam and sampling, giving optimal growth and higher yield.

A palindromic DNA sequence reads the same on both strands when each is read in the 5′→3′ direction. Examples (recognition sites of restriction enzymes):

• EcoRI: 5′-GAATTC-3′
• BamHI: 5′-GGATCC-3′
• HindIII: 5′-AAGCTT-3′
• PstI: 5′-CTGCAG-3′
• SmaI: 5′-CCCGGG-3′

To create one: write a sequence and then its reverse complement next to it, e.g. take 5′-GCA, its reverse complement is TGC, giving 5′-GCATGC-3′; the other strand read 5′→3′ is also GCATGC.

EcoRI GAATTC, BamHI GGATCC, HindIII AAGCTT, PstI CTGCAG, SmaI CCCGGG.

Natural recombinant DNA forms during crossing over between non-sister chromatids of homologous chromosomes in pachytene of prophase I of meiosis, when segments are exchanged.

During crossing over in pachytene of prophase I.

A reporter gene codes for an enzyme whose activity is easy to see, e.g. β-galactosidase (lacZ). The foreign DNA is inserted inside the reporter gene (insertional inactivation).

• Cells that took up a plasmid without an insert have an active β-galactosidase and give blue colonies on a medium with a chromogenic substrate (X-gal).
• Cells with the recombinant plasmid have the reporter gene inactivated and give colourless (white) colonies.

Thus the colour distinguishes recombinants from non-recombinants without having to grow them on two antibiotic plates. (Green fluorescent protein and luciferase are other reporters.)

The insert inactivates the reporter (e.g. β-galactosidase): recombinants form white colonies, non-recombinants blue (with X-gal).

• (a) Origin of replication (ori): the DNA sequence where replication starts. Any DNA linked to it can be replicated in the host cell; it also controls the copy number of the linked DNA.
• (b) Bioreactors: large vessels (100–1000 L) in which raw materials are converted into products by microbes, plant, animal or human cells under controlled conditions (temperature, pH, oxygen, nutrients). The stirred-tank type is most common.
• (c) Downstream processing: the processes after biosynthesis to make the product ready for use: separation and purification of the product, followed by formulation with preservatives and quality control (clinical trials for drugs).

(a) Sequence where replication starts. (b) Large controlled vessels for producing products from cells. (c) Separating, purifying and formulating the product.

• (a) PCR (polymerase chain reaction): a technique to make many copies of a specific DNA segment in vitro, using two primers and a heat-stable DNA polymerase (Taq polymerase). Each cycle has three steps: denaturation (high heat separates strands), annealing (primers bind) and extension (polymerase builds new strands). About a billion copies can be made in 30 cycles.
• (b) Restriction enzymes and DNA: restriction endonucleases recognise specific palindromic base sequences in DNA and cut both strands there, often giving sticky ends. They are "molecular scissors" used to cut DNA for making recombinant DNA.
• (c) Chitinase: an enzyme that breaks down chitin; it is used to break the cell walls of fungi to release their DNA during DNA isolation.

(a) In vitro DNA amplification by repeated denaturation, annealing and extension. (b) Enzymes that cut DNA at specific palindromic sites. (c) Enzyme that digests fungal cell walls.

(a) Plasmid DNA: small, circular, extra-chromosomal DNA in bacteria that replicates on its own; carries non-essential genes (e.g. antibiotic resistance); used as a vector. Chromosomal DNA: the main, large DNA of the cell carrying the essential genes.

(b) RNA: ribose sugar, uracil, usually single-stranded. DNA: deoxyribose sugar, thymine, double-stranded helix.

(c) Exonuclease: removes nucleotides from the ends of DNA. Endonuclease: cuts at specific positions within the DNA (e.g. restriction enzymes).

(a) Small, circular, extra copy vs the main genome. (b) Ribose and U, single strand vs deoxyribose and T, double helix. (c) Cuts from the ends vs cuts inside.