Preparing an Overnight Culture of Escherichia coli Laboratory 6

Overview: Purpose: From LB/amp/ara plate: 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 pARA-R plasmid Inoculate an overnight culture

Introduction Gene for mFP originally isolated from sea anemone Protein can fuse to other proteins – used in research pARA-R major control elements: Required to express the rfp gene 1. araC gene Codes for AraC 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 araC gene PBAD rfp gene Transcription mRNA Translation araC protein

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

RFP expression Bruce Wallace araC protein prevents RFP transcription by causing a loop to form in the region of the fp gene r araC gene araC protein PBAD rfp gene

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

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

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

What will you need to do? Tell students to work quickly to avoid contamination Discuss in detail rfp expression AraC protein involved with turning arabinose operon ON and OFF!! Binds to promoter OR arabinose Need 2.5 mL 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 mFP-expressing colony (red) Into flask containing LB/amp/ara broth Flask placed on shaker Shaken overnight Encourages cell division and rfp expression

Colony isolation and culture 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 mFP Ready to be lysed and purified!