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!