1. insertional inactivation of antibiotic resisteance gene
1.1. lacZ: IPTG inducible vector
1.1.1. blue-white screening
1.1.1.1. lacZ part in the plasmid can insert with new DNA
1.1.1.1.1. blue contain beta galactosidase because still contain enzyme to break down the algae into blue beta-galactosidase.
1.1.1.1.2. white contain recombinant plasmid because lacZ is cutted from the plasmid. enzyme no longer active. no blue colouration from beta galactosidase release.
1.1.2. cloning
1.1.2.1. multiple choning site:need to digest plasmid and insert gene in interest.
1.1.2.2. contain sticky end, isolate plasmid and insert to the sticky end and produce recombinant plasmid.
1.1.2.3. isolate with electrophoresis gel and estimate the size to check whether the gene of interest exist: two bands with shorted gene of interest and longer plasmid.
1.2. Cloning into pBR322 in the BamHI site
1.2.1. Theory:Original plasmid contain ampicillin resistance and tetracycline resistance.
1.2.1.1. Tetracycline resistance of the original plasmid replaced by inserted DNA. recombinant plasmid or cell is resistance to ampicillin but not tetracycline
1.2.1.2. original plasmid with no transformation resistance to both.
1.2.2. screening
1.2.2.1. after transformation, grow in ampicilin medium, incubated until colony appear
1.2.2.1.1. all transformed cell is resistance to ampicillin so can survive and grow
1.2.2.1.2. non-transformed cell has no resistance so died.
1.2.2.2. after that, put in the algae medium contain tetracycline
1.2.2.2.1. colony that grow has tetracycline resistance, so is not transformed cell
1.2.2.2.2. colony that no grow on algae does not have resistance to tetracycline, so it is transformed cell
1.3. New node
2. expression vector
2.1. purpose:
2.1.1. produce large amount of protein for biochemical
2.1.2. examine the function of protein
2.2. criteria
2.2.1. multicopy plasmids
2.2.2. multiple selective marker
2.2.3. strong and different promoter
2.2.4. regulatable promoter
2.3. fusion protein
2.3.1. recombinant protein that consist short peptide
2.3.2. advantage
2.3.2.1. increase stability
2.3.2.2. allow transportation
2.3.2.3. aid purification
2.3.2.3.1. agarose beat bound with glutathione via the glutathione-5-transferase compound, not being washed out. Then elute with glutathione, to gain pure protein
2.3.2.3.2. His tag and Ni on agarose : equilibration by Ni, binding with his tag and elution with imidazole