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Transformation of E.coli with pGLO

Transformation of E.coli with pGLO. Dan Prochaska and Jeff Winslow Talawanda/Butler Tech. Purpose. Challenge students to participate in cutting edge laboratory activities that emphasize important skills carried out in a structured environment.

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Transformation of E.coli with pGLO

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  1. Transformation of E.coli with pGLO Dan Prochaska and Jeff Winslow Talawanda/Butler Tech

  2. Purpose • Challenge students to participate in cutting edge laboratory activities that emphasize important skills carried out in a structured environment. • To make connections to modern commercially viable protocols, i.e. human insulin production.

  3. Lesson Plan • Three day lesson. • Day 1: Powerpoint introduction, handouts with protocol, streaking starter plates, and pre-assessment • Day 2: Complete transformation protocol, student groups make predictions about results • Day 3: Observe plates using U.V. pen lights and record observations. Make connections to real world applications. Complete post assessments.

  4. pGLO lab. Objectives • Transform E. coli host cells with plasmid DNA. • Predict the results on selective agar plates. • Observe and record actual plate data. • Draw conclusions from the experiment and list possible sources of error.

  5. Introduction What is genetic transformation? • When host bacteria acquire novel traits that are encoded in genes contained on plasmid, (pGLO) DNA. What new traits will E.coli acquire from the transformation? Antibiotic resistance (ampicillin) Green Fluorescence: Where did this trait come from?

  6. What is the source of green flourescence? • The marine jellyfish Aequorea victoria

  7. Plasmid DNA • Very small (103bp) piece of circular DNA • Replicates autonomously, (ori site). • Is in addition to the large (106bp) E. coli chromosome. • Can be exchanged between cells. • Carries important genes for traits such as antibiotic resistance. • In this laboratory we are investigating ampicillin resistance.

  8. Plasmid DNA A small circular piece of DNA that is naturally occurring in some bacteria. It can be exchanged between cells and often carries antibiotic resistance genes

  9. pGLO an engineered plasmid

  10. pGLO plasmid genes • GFP gene encodes a green fluorescent protein (GFP) • Bla gene encodes the enzyme beta lactamase that degrades the antibiotic ampicillin thus, protecting the cell from its harmful effects. • AraC is a regulatory gene that encodes a protein that acts like a switch that turns on the expression of the GFP gene.

  11. Transformation- Terms • Competence: cells are able to take up a plasmid from their surroundings • How do we make our E.coli cells competent? • Gene expression: cells replicate, transcribe, and translate genes located on the pGLO plasmid

  12. Transformation Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids

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