rpARA Recombinant mFP Expression Sequence

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Biotechnology Lab Program
Bruce Wallace
pARA-R Sequence
RFP Expression Sequence
Laboratory Protocols by
Marty Ikkanda
Powerpoint by
Anthony Daulo Kristi Schramm
Pierce College, Woodland Hills, CA
V.1.2a.2
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pARA-R construct
Bruce Wallace
Recombinant plasmid of interest
pARA-R
4720 bp
BamH I
rfp 702bp
Hind III
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Engineering the Plasmid ligation of rfp gene
into p-ARA
Bruce Wallace
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pARA-R construct
Bruce Wallace
Recombinant plasmid of interest
pARA-R
4720 bp
BamH I
rfp 702bp
Hind III
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(No Transcript)
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RFP expression
Bruce Wallace
Transcription
Translation
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RFP expression
Bruce Wallace
araC protein
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RFP expression
Bruce Wallace
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
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Preparing competent cells for transformation
Bruce Wallace
Lipid bilayer (inner)
Peptidoglycan layer
Adhesion zone
Lipid bilayer (outer)
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Transforming Escherichia coli with pARA-R
Bruce Wallace
Recombinant Plasmids
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Transforming Escherichia coli with pARA-R
Bruce Wallace
Lipid bilayer (inner)
Peptidoglycan layer
Adhesion zone
Lipid bilayer (outer)
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Growth of transformed bacteria on various plates
Bruce Wallace
P plates
LB
LB/amp
LB/amp/ara
P- plates
No growth
LB
LB/amp
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Preparing an overnight culture of E. Coli for RFP
expression
Bruce Wallace
Colony isolation and culture
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Bruce Wallace
RFP
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Bruce Wallace
RFP
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Purification of RFP from an overnight culture
Bruce Wallace
Overnight culture
Cell pellet with RFP
Lysed cells
Pellet cell debris
RFP with binding buffer