Principles of genetic engineering

OBJECTIVE To describe the main stages in genetic engineering

What is genetic engineering Also known as recombinant DNA technology, –altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. Could involve –inserting a foreign gene from one species into another, forming a transgenic organism –altering an existing gene so that its product is changed –changing gene expression so that it is translated more often or not at all.

Purpose of GMOs Improved feature –Herbicide resistance gene inserted into a plants’ genome For synthesis of useful products –Human hormones mass-produced in bacteria insulin, growth hormone –  -carotene in rice grains turns into vitamin A when eaten

Examples of transgenics Extended shelf-life tomato Herbicide resistant soybean, corn and canola Insect resistant cotton and corn NormalTransgenic Virus resistant papaya Cows with enzyme to increase milk production

Basic steps in genetic engineering 1.Isolate the gene 2.Insert into a vector 3.Insert modified vector into host cell (Transformation & selection) 4.Allow host to multiply and to synthesise protein 5.Separate and purify the product of the gene

Step 1: Isolating the gene A.Using restriction enzymesUsing restriction enzymes 1.A probe is used to locate the gene 2.Restriction enzymes recognise restriction sites and cut out gene Restriction sites are palindromic –The same sequence when read left to right (5’ to 3’) on one strand and right to left on complementary strand EcoR1 produces sticky ends Sma1 produces blunt ends

Restriction enzymes Originally obtained from bacteria Cuts up phage DNA DNA of virus that infect bacteria Why is this useful? Why are the staggered cuts called sticky ends? Terminal transferase enzyme can add sticky ends if restriction enzyme generates blunt ends

Step 1: Isolating the gene B.Using Reverse transcriptaseUsing Reverse transcriptase Gene for this enzyme originally found in retroviruses (contain RNA instead of DNA) –why is the enzyme useful for these? Converts mRNA into single-stranded cDNA –E.g. insulin mRNA from B-cells of islets of Langerhans Then DNA polymerase produces complementary strand to form double stranded DNA Advantage – more mRNA in cell than DNA Why is it an advantage to use cDNA if you are inserting a eukaryotic gene into a prokaryote?

Step 1: Isolating the gene C.Using an automated sequencer Amino acid sequence of protein analysed Gene for protein synthesised Using triplet code

Step 2: Inserting gene into vectorInserting gene into vector Vector – molecule of DNA which is used to carry a foreign gene into a host cell, e.g. Bacterial plasmids –double stranded circular DNA Virus genomes –Can carry large fragments Yeast cell chromosomes

Recombinant DNA DNA from different sources That have been combined

Plasmid and foreign DNA are cut with the same enzyme –Generates complementary sticky ends that can anneal Ligase enzyme seals gaps/nicks in S-P backbone –forms phosphodiester bonds between inserted gene and the plasmid

Step 3: inserting vector into host Transformation

1.Soak E.coli in CaCl, mix with plasmid, mild heat shock –Makes membrane more permeable to plasmids 2.Electroporation –High voltage pulse disrupts membrane 3.Microinjection –using fine micropipette

Transformation 4.Viral infection –using virus’ own mechanism to insert DNA 5.Bacterial infection –Agrobacterium tumefaciens which naturally insert Ti plasmids into plant genome 6.Liposomes –containing DNA easily cross lipid membrane

Selection Colonies of bacteria will grow from each bacterial cell which –Did not take up plasmid –Took up plasmid which re-sealed without inserted gene –Transformed bacteria Those that took up recombinant plasmids (have inserted gene) How can we tell the difference?

Selection Transformed cell Cell with plasmid resealed without insert Cell with its usual plasmid Purple represents inserted gene

Selection methods Using antibodies for protein produced Adding a fluorescent marker gene to plasmid –Glowing bacteria have plasmid Replica plating

Selection by replica plating Using plasmids with 2 genes for antibiotic resistance

Selection by replica plating Plasmid used carries –ampicillin resistance gene (amp) Allows transformed bacteria to grow in agar plates with ampicillin antibiotic (‘amp plates)’ –Tetracyclin resistance gene (tet) Gene is inserted within this gene Tet gene inactivated Transformed bacteria cannot grow in ‘Tet plates’

Selection by Replica plating Those that grow in amp plates but not in tet plates are transformed bacteria

Step 4: Multiplication of the host cells by cloning Large scale fermenters Bacteria undergo binary fission –Large numbers produced quickly –E. coli divide every 20 min –All genetically identical because of asexual reproduction Transcription and translation

Step 5: Extraction & purification of desired gene product Bacteria killed and separated from proteins by centrifugation Protein of interest separated from others by Large scale chromatography ultrafiltration

Try the paper simulation to help you understand how the process works!paper simulation