Laboratory 6

Preparing an Overnight Culture of Escherichia coli

Overview:

Purpose: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 plasmidInoculate 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 gene1. araC gene

Codes for AraC protein, which helps turn rfp gene on and offActs as a repressor by blocking promoter

2. promoter region (PBAD)Portion of DNA where regulation of rfp expression

occursRNA polymerase binds

3. rfp location

RFP expression

araC gene rfp genePBAD

Transcription

mRNA

Translation

araC protein

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Introduction

2. promoter region (PBAD)If no arabinose:

AraC protein physically bind to plasmidAt promoter siteAt region near the araC

geneDNA bends around and

blocks transcription cannot occur

RNA polymerase cannot bind to promoter site

Bacterium cannot produce mFP

RFP expression

rfp genePBAD

araC protein

araC gene

araC protein prevents RFP transcription by causinga loop to form in the region of the fp gener

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Introduction2. promoter region (PBAD)

If arabinose present:Arabinose binds with AraC

protein Prevents DNA loop from

formingRNA polymerase can join

complex after conformational change

Three molecule complex binds promoter site on DNA

DNA transcribes rfp gene to produce mRNATranslates to mFP!!Stored as inclusion bodies

RFP expression

araC protein

arabinose

araC gene rfp genePBAD

arabinose – araC proteincomplex

RNA polymerase

Arabinose – araC protein complex prevents DNA loopingand helps to align RNA polymerase

on the promoter site (PBAD).

mRNA

Transcription

TranslationRFP

(red fluorescentprotein)

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MaterialsMaterials:

LB/amp/ara plate (from Lab 5)

Sterile flask with LB/amp/ara broth

Equipment & Supplies:ShakerPlastic inoculating

loops

What will you need to do?Tell students to work quickly to avoid

contaminationDiscuss 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 groupUse sterile loop to transfer small sample of cells

to LB/amp/ara brothShake culture 1-2 days at room temperature

MethodsUse a clean plastic inoculating loop to

transfer a few cellsFrom mFP-expressing colony (red)Into flask containing LB/amp/ara broth

Flask placed on shaker Shaken overnightEncourages cell division and rfp expression

Colony isolation and culture

Preparing an overnight culture of E. Coli for RFP expression

LB/amp/arabroth

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Conclusions

Cells grown that have been expressing mFPReady to be lysed and purified!