Preparing an Overnight Culture of Escherichia coli Laboratory

Preparing an Overnight Culture of Escherichia coli Laboratory 6

Do Now How does the information of this unit connect to the rest of our content in AP bio? Will there be any questions about this in the AP exam?

Objectives: Start a bacterial culture that will produce the mutant fluorescent protein This will lead to the isolation and purification of that protein From LB/amp/ara plate: Will select one red colony that has been transformed with p. ARA-R plasmid Inoculate an overnight culture

Introduction Gene for m. FP originally isolated from sea anemone Protein can fuse to other proteins – used in research p. ARA-R major control elements: Required to express the rfp gene 1. ara. C gene Codes for Ara. C protein, which helps turn rfp gene on and off Acts as a repressor by blocking promoter 2. promoter region (PBAD) Portion of DNA where regulation of rfp expression occurs RNA polymerase binds 3. rfp location

RFP expression Bruce Wallace PBAD ara. C gene Transcription m. RNA Translation ara. C protein rfp gene

Introduction 2. promoter region (PBAD) If no arabinose: Ara. C protein physically bind to plasmid At promoter site At region near the ara. C gene DNA bends around and blocks transcription cannot occur RNA polymerase cannot bind to promoter site Bacterium cannot produce m. FP

RFP expression Bruce Wallace ara. C protein prevents RFP transcription by causing a loop to form in the region of the rfp gene ara. C protein PBAD rfp gene

Introduction 2. promoter region (PBAD) If arabinose present: Arabinose binds with Ara. C protein Prevents DNA loop from forming RNA polymerase can join complex after conformational change Three molecule complex binds promoter site on DNA transcribes rfp gene to produce m. RNA Translates to m. FP!! Stored as inclusion bodies

RFP expression Bruce Wallace arabinose Arabinose – ara. C protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site (PBAD). RFP (red fluorescent Translation protein) RNA polymerase ara. C– protein arabinose ara. C protein complex m. RNA Transcription ara. C gene PBAD rfp gene

Materials: Equipment & Supplies: LB/amp/ara plate Shaker (from Lab 5) Sterile flask with LB/amp/ara broth Plastic inoculating loops

What will you need to do? Tell students to work quickly to avoid contamination Discuss in detail rfp expression Ara. C protein involved with turning arabinose operon ON and OFF!! Binds to promoter OR arabinose Need 2. 5 m. L overnight culture for each group Use sterile loop to transfer small sample of cells to LB/amp/ara broth Shake culture 1 -2 days at room temperature

Methods Use a clean plastic inoculating loop to transfer a few cells From m. FP-expressing colony (red) Into flask containing LB/amp/ara broth Flask placed on shaker Shaken overnight Encourages cell division and rfp expression

Preparing an overnight culture of E. Coli for RFP expression Bruce Wallace Colony isolation and culture LB/amp/ara broth

Conclusions Cells grown that have been expressing m. FP Ready to be lysed and purified!