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530L_notes_lecture5 - Lecture 5 IX Protein expression A...

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Lecture 5 IX. Protein expression A. Strains of bacteria 1. Characteristics of cloning strains: Ex. DH5alpha Have been genetically modified to uptake and replicate exogenous DNA (plasmid). Majority of endogenous restriction endonucleases have been deleted because we do not want to degrade the piece of DNA that we inserted. 2. characteristics of expression strains: Ex. Bl21:DE3-origami Optimized strain of Ecol-I cells that are genetically manipulated to over-express the clone gene on our plasmid. B. Why pET21? 1. “molecular machinery”: Possesses a number of engineered pieces of molecular machinery that allows us to make lots and lots of proteins. Everything we need is already engineered in pET21c. LOOK AT VECTOR MAP MCS: looking at coding strand 5’ 3’ The region where we pasted our gene. The molecular machineries: 1. T7 Promoter : Specific DNA sequence where T7 RNA polymerase binds. A member of T7 phage engineered into the pET21c. 2. LacO (lac operator): DNA sequence that is bound by or to which lac repressor binds. We will use this ON/OFF characteristic of the lac operator/repressor. 3. RBS (ribosomal binding site) DNA element that is expressed in the mRNA.
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Component of mRNA near the 5’ end, upstream of the initiation codon, where it is bound by the ribosome for translation. 4. NdeI site : CATATG : this ATG is the initiation codon! Met- By placing the Aphos gene right after the NdeI gives a good proximity to the RBS such that the gene is transcribed right after the ATG codon. Perfect proximity from the promoter and RBS to be transcribed properly. 5. T7 terminator : T7 polymerase binds to T7 promoter, transcribe mRNA that includes the RBS, and from NdeI to XhoI (which includes the Aphos gene) within the MCS and transcribe until it hits the terminator . ***All molecular goodies to express/turn on are already engineered into pET21.
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530L_notes_lecture5 - Lecture 5 IX Protein expression A...

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