BIO 325L Lecture 9 - This week in lab 1 Quiz GMO results...

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This week in lab Quiz GMO results Bacterial transformation results Protein isolation (GFP) Mini-practical: PCR prep and pipetting 1
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What do you need to turn in? Tonight (3/26) is deadline for midterm re- grade requests Homework 8 (crossing over Chi-square analysis) Homework 9 2
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Today in lecture Protein purification and GFP 3
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Bacterial expressed GFP Last week: Genetic transformation of E. coli Inoculation of agar plates Transformed bacteria produce GFP This week: Purification of GFP Liquid cultures of transformed bacteria GFP protein is purified via column chromatography 4
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LB/Amp LB/Amp/Ara LB LB/Amp LB/Amp/Ara LB Gene selection Grow transformed bacteria and control bacteria under various conditions Bacteria containing the gene of interest are selected for, they will grow ( screening is a different process) Control - - + Transformed + + + On which of the 6 plates will there be bacterial growth? 5
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Gene selection Which plate should have fluorescent colonies? LB/Amp LB/Amp/Ara LB 6
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Isolating proteins Proteins are small and only a fraction of a cell Isolating proteins very broadly involves two processes: Purification Cells are composed of many different macromolecules Need to isolate protein of interest from all, or at least most, other molecules Concentration A protein of interest is likely a small % of a cell’s volume 7
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Three common steps of isolation 1. Bacterial concentration and lysis If the bacteria contain your protein of interest, you can concentrate your protein by concentrating the cells! 2.
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BIO 325L Lecture 9 - This week in lab 1 Quiz GMO results...

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