Transcript Preparing an Overnight Culture of Escherichia coli

Preparing an Overnight Culture of
Escherichia coli
Laboratory 6
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
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
promoter region (PBAD)
Portion of DNA where regulation of rfp expression occurs
RNA polymerase binds
rfp location

 2.

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 3.
RFP expression
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araC gene
PBAD
Transcription
mRNA
Translation
araC protein
rfp gene
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
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araC protein prevents RFP transcription by causing
a loop to form in the region of the fpr gene
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
RFP expression
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arabinose
Arabinose – araC protein complex prevents DNA looping
and helps to align RNA polymerase
on the promoter site (PBAD).
RFP
(red fluorescent
Translation
protein)
RNA polymerase
araC– protein
arabinose
araC protein
complex
mRNA
Transcription
araC gene
PBAD
rfp gene
Materials
 Materials:
 Equipment & Supplies:
 LB/amp/ara plate (from
 Shaker
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
 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
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!