Bio 203 9-18-08 review of transformation objectives discussion of measuring transformation efficiency overview of next series of labs Objectives of transformation labs 1. Illustration of the ?central dogma???genotype? to ?phenotype? transformed bacteria are now resistant to antibiotic ampicillin-how? bla gene on plasmid encodes a protein enzyme (beta lactamase) that breaks down ampicillin under appropriate conditions (see below), bacteria also glow due to expression of a green fluorescent protein (GFP) 2. Illustration of gene regulation, specifically at level of transcription using recombinant DNA, the jellyfish GFP gene was linked to the promoter sequence of the arabinose operon, this DNA was inserted into a plasmid (subsequently named pGLO) in response to arabinose, bacteria containing pGLO transcribe mRNA for GFP, & following translation of that mRNA? Bacterial chromosomal DNA containing thousands of genes encoding proteins required for growth and reproduction (some constitutively expressed, others regulated) GFP, regulated expression: GFP gene transcribed only in presence of arabinose; mRNA translated into GFP araC, constitutive expression: araC gene transcribed into mRNA; mRNA translated into araC protein, a ?transcription factor? beta lactamase, constitutive expression: bla gene transcribed into mRNA; mRNA translated into beta lactamase, an enzyme that provides resistance to ampicillin Where do we go from here? GFP purification ? a scenario ?clone? a gene of interest encoding our ?target? protein Link target gene to arabinose operon promoter and GFP gene Insert above into plasmid, transform bacteria Grow bacteria to large number, and activate expression Isolate all protein from bacterial cells, then purify target-GFP
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