pARA-R Sequence
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pARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce College, Woodland Hills, CA V.1.2a.2 Bruce Wallace
description
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. pARA-R construct. Bruce Wallace. Recombinant plasmid of interest. - PowerPoint PPT Presentation
Transcript of pARA-R Sequence
pARA-R SequenceRFP Expression Sequence
Biotechnology Lab Program
Laboratory Protocols by:
Marty IkkandaPowerpoint by:
Anthony DauloKristi Schramm
Pierce College, Woodland Hills, CAV.1.2a.2
Bruce Wallace
pARA-R construct
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
Bruce Wallace
Engineering the Plasmid: ligation of rfp gene into p-ARA
sticky endBamH I
sticky endHind III
sticky endBamH I
sticky endHind III
Bruce Wallace
pARA-R construct
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
Bruce Wallace
RFP expression
araC gene rfp genePBAD
Transcription
mRNA
Translation
araC protein
Bruce Wallace
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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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
Translation
RFP(red fluorescent
protein)
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Preparing competent cells for transformation
Lipid bilayer(inner)
Lipid bilayer(outer)
Peptidoglycanlayer
Adhesion zone
Calcium ions
Bruce Wallace
Transforming Escherichia coli with pARA-R
Recombinant Plasmids
Competent Cells
pARA-R
Bruce Wallace
Calcium ions
pARA-R
Transforming Escherichia coli with pARA-R
Adhesion zone
Lipid bilayer(inner)
Lipid bilayer(outer)
Peptidoglycanlayer
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Growth of transformed bacteria on various plates
P+ plates
P- plates
LB LB/amp LB/amp/ara
LB LB/amp
No growth
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Colony isolation and culture
Preparing an overnight culture of E. Coli for RFP expression
LB/amp/arabroth
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RFP
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Purification of RFP from an overnight culture
Overnightculture
Cell pelletwith RFP
Lysedcells
Pelletcell debris
RFP withbinding buffer
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