Biotechnology: Principles and Processes

Modern biotechnology rests on two core techniques: genetic engineering, which alters the chemistry of genetic material, and bioprocess engineering, which maintains sterile conditions to grow large quantities of the desired microbe or cell. In this chapter you will learn how Stanley Cohen and Herbert Boyer created the first recombinant DNA in 1972 by linking an antibiotic resistance gene to a plasmid of Salmonella typhimurium, and you will study the tools of recombinant DNA technology: restriction enzymes such as EcoRI that recognise palindromic sequences and produce sticky ends, DNA ligase, cloning vectors such as pBR322 with an origin of replication, selectable markers and cloning sites, and competent hosts. The chapter then explains the process step by step: isolation of DNA, cutting at specific sites, separating fragments by agarose gel electrophoresis, amplifying genes by the polymerase chain reaction using Taq polymerase, ligation, and transformation by heat shock, microinjection or biolistics. Finally, you will learn about selection of recombinants by insertional inactivation, and large-scale production in stirred-tank bioreactors followed by downstream processing.

Detailed Worksheet: Biotechnology: Principles and Processes Section A - Definitions (10 marks) 1. Define biotechnology according to the European Federation of Biotechnology. Name the two core techniques that enabled modern biotechnology. (2 marks) 2. What are restriction endonucleases? Explain the naming of EcoRI. (2 marks) 3. What is a palindromic nucleotide sequence? Write the palindrome recognised by EcoRI with its 5' and 3' ends. (2 marks) 4. Name any three features required to facilitate cloning into a vector. (2 marks) 5. What is downstream processing? Why is it necessary before a product is marketed? (2 marks) Section B - Calculations and Applications (15 marks) 6. A restriction enzyme recognises a specific 6 base pair sequence. Assuming all four bases occur randomly and equally, calculate the average distance (in base pairs) between two sites. How many fragments would you expect when a linear DNA of 40,960 bp is cut? Repeat for a 4 base pair recognition sequence. (3 marks) 7. A linear DNA molecule has three EcoRI sites and a circular plasmid has three EcoRI sites. How many fragments are produced in each case on complete digestion? Explain the difference. (3 marks) 8. A PCR begins with a single double-stranded DNA template. Calculate the number of DNA copies after 25 cycles assuming perfect doubling (take 2^10 = 1,024). State the three steps of each cycle and the temperature at which denaturation occurs. (3 marks) 9. A plasmid of 4,361 bp is cut by an enzyme at a single site and a foreign gene of 1,200 bp is inserted. What is the size of the recombinant plasmid? On a gel, would the recombinant plasmid DNA (linearised) move faster or slower than the linearised vector alone? Explain. (3 marks) 10. In pBR322, a foreign gene is inserted into the BamHI site located in the tetracycline resistance gene. Predict the growth of (i) non-transformed cells (ii) cells with the non-recombinant plasmid (iii) cells with the recombinant plasmid on ampicillin medium and on tetracycline medium. (3 marks) Section C - Diagrams (10 marks) 11. Draw a labelled diagram of the E. coli cloning vector pBR322 showing the restriction sites (HindIII, EcoRI, BamHI, SalI, PvuII, PstI, ClaI), ori, rop, and the ampR and tetR genes. (4 marks) 12. Draw a labelled diagram showing the action of EcoRI on DNA, producing sticky ends, and the joining of a foreign DNA fragment with a vector by DNA ligase. (3 marks) 13. Draw a labelled diagram of a simple stirred-tank bioreactor showing the motor, foam breaker, sterile air inlet (sparger), pH, temperature and acid-base controls, and the flat-bladed impeller. (3 marks) Section D - Analysis and Higher-order Thinking (15 marks) 14. A student isolates DNA from plant leaves. Explain the role of each in the procedure: cellulase, ribonuclease, protease and chilled ethanol. How would the enzymes differ if the source were a fungus or a bacterium? (5 marks) 15. Selection of recombinants using antibiotic markers requires two plates, but the blue-white screening method uses only one. Explain insertional inactivation in both methods and analyse why the chromogenic substrate method is more convenient. (5 marks) 16. Explain why bacteria must be made competent before transformation, and describe the calcium chloride and heat shock method. Compare it with microinjection and biolistics, stating which method is suitable for plant cells and for animal cells. (5 marks) Instructions: Time allowed 2 hours. Attempt all sections. Draw all diagrams neatly in pencil and label every part. Show working for all calculations.