Lessons 30–31 · 2 lessons · A. G‘afurov, A. Abdukarimov, J. Tolipova, O. Ishankulov, M. Umaraliyeva, I. Abdurahmonova. Biology, Grade 10, 1st edition. “Sharq” publishing and printing joint-stock company, Tashkent, 2017
31
Obtaining recombinant DNA
Textbook: pp. 134–135
GoalExplain the steps of obtaining recombinant DNA (obtaining the gene, vector, cutting and joining, introduction into the cell, cloning and selection).
Recombinant DNA is an artificial combination of DNA pieces from different sources. It was first obtained in 1972–73 in the experiments of P. Berg, S. Cohen and H. Boyer. The steps: 1) obtain the needed gene from the donor organism – by cutting with a restriction enzyme, by making cDNA from mRNA with revertase, or, if its nucleotide sequence is known, by chemical synthesis; 2) choose a vector – usually a plasmid (or a virus or phage) that carries an antibiotic-resistance gene; 3) cut gene and vector with the same restriction enzyme and join them with ligase – a recombinant plasmid forms; 4) introduce it into a recipient cell (E. coli, yeast) – transformation; 5) selection – if antibiotic is added to the medium, only bacteria that took up the recombinant plasmid survive; 6) cloning – such cells multiply, the copies of the foreign gene multiply (gene cloning) and the gene makes protein (expression). Human insulin, growth hormone and interferon are made this way. Gene therapy, replacing a faulty gene with a healthy one, is being studied.
Worked examples
The insulin gene (cDNA) is joined into a plasmid and put into E. coli. If ampicillin is added to the medium, only bacteria with a plasmid carrying the ampicillin-resistance gene grow; insulin protein is obtained from these colonies.
If one recombinant bacterium divides every 30 minutes, in 5 hours it divides 10 times and gives 2¹⁰ = 1024 cells – 1024 copies of the foreign gene.
Class activity
“Recombinant builder”: groups cut paper plasmid and gene cards (an adult helps with scissors), join them in the right order and arrange the 6 steps.
Practice
1
List the steps of obtaining recombinant DNA in order.
Obtain gene – choose vector – cut and join with ligase – introduce into the cell – select – clone and express.
2
Why are the gene and vector cut with the same restriction enzyme?
The same sticky ends form, and being complementary they join.
3
A bacterium divides every 20 minutes. How many cells does one give in 1 hour 40 minutes? (Number only.)
32
4
Why is an antibiotic used to select recombinant bacteria?
The vector carries a resistance gene: only bacteria that took up the plasmid survive with the antibiotic; the others die.