6 Steps in in vivo cloning In vitro to start withStage 1Stage 2Stage 3Stage 4
7 Stage 1 – obtaining the DNA for the gene we want to clone, we looked at this last week Stage 2 – is insertion of the DNA into a vectorA vector is a carrier and is required in order to transfer the extracted gene into a chosen bacterial cell.An example of a vector is a plasmid i.e. a circular piece of DNA found in a bacterium.
8 How do we insert the gene into the plasmid How do we insert the gene into the plasmid? (refs p 6 booklet p A2 book)The plasmid is treated with the same restriction endonuclease enzyme as the extracted DNA.This means it has complementary sticky ends to the extracted DNA so they can be joined together by the enzyme DNA ligase.
9 Stage 3 – inserting the plasmid into bacterial cells Bacteria and plasmids are mixed in a medium containing calcium ions.This makes the bacterial cell wall more permeable to plasmids, which pass into the cytoplasm of the bacteria.Bacteria which contain the recombinant plasmid are said to be transformed
10 Try this exercise with your pieces of human and plasmid DNA (You will need to remember the recognition site for ECoR1. )Treat the human DNA with Eco R1 restriction endonuclease enzyme ( )Use your Eco R1 restriction enzymei.e to cut the plasmid and produce sticky ends complementary to your human gene.Use your DNA ligase i.e To join the sticky ends of the plasmid with those of the human geneYou have now made a recombinant plasmid!!
11 Adenoviruses and liposomes are other examples of vectors, apart from plasmids
12 Steps in in vivo cloning In vitro to start withStage 1Stage 2Stage 3Firstly, we need to identify which bacteria contain the recombinant plasmidsStage 4
13 Identifying Transformed Host Cells These are cells that have taken up the plasmidUse antibiotic resistanceGene for antibiotic resistance is found in some plasmids, so insert required gene into themGrow bacteria on medium containing antibioticOnly those bacteria that have taken up the plasmid will survive!
15 How can the transformed bacteria be identified? By culturing the bacteria on an agar plate which contains the antibiotic.Untransformed bacteria (no resistance)Transformed bacteria (contain the resistance gene)NO GROWTHGROWTH
16 Now make sure you have completed all the qs on page 6
17 How can we identify bacteria which have taken up both the gene and the plasmid? (page 7) We use GENE MARKERS. These identify both the presence of the plasmid AND the presence of the required gene.3 types of gene marker:Antibiotic resistanceFluorescenceAn enzyme
18 Recombinant plasmid produced: Use plasmids with 2 resistance genes (getting complicated and clever now!)Will the tetracycline resistance gene still function?Ampicillin resistancegeneHuman gene inserted into tetracycline resistance geneRecombinant plasmid produced:
19 But....Sometimes the original plasmid rejoins its sticky ends to itself without inserting the foreign gene
20 So..we need to ensure we only select bacteria with the recombinant plasmid In this example – we first grow the bacteria on a plate containing ampillicinOnly bacteria with plasmids containing this resistance gene will growNow grow the surviving bacteria on the second antibiotic – usually TetracyclineWhat will happen?This is pointless so what to do?
21 Replica PlatingTake small samples of bacteria surviving the first antibioticGrow on 2nd antibioticNote positions of dead coloniesThese are the ones containing the required gene
22 Fluorescent MarkersInsert gene for fluorescence (from a fish) into plasmid.Insert required gene into the middle of it and put into bacteriaGrow bacteriaBacteria that have taken up the gene and the plasmid will NOT fluoresce
23 Finally...Stage 4 Growth / cloning (in vivo) The transformed bacteria, which have now been identified, are now grown on a large scale in a fermenter. They will secrete the gene product (e.g. insulin) into the medium they are growing in and it can then be extracted and purified.
24 Now test yourself...Complete the summary questions on page 253 of the text bookAnswer the exam question on page 25 and 26 of your booklet.