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pARA-R RecombinantRFP Expression Sequence
biotechnology lab program
Laboratory Protocols by:
Marty IkkandaPowerpoint by:
Anthony DauloPierce College, Woodland Hills, CA
V.1.2.1
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restriction analysis of pKAN-R and pARA
pKAN-R
pARA
BamH I
Hind III
BamH IHind III
rfp702 bp
5408 bp
4058 bp
40 bp
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restriction analysis of pKAN-R and pARA
BamH I Hind III
702bp
40 bp
BamH I Hind III
BamH IHind III
BamH IHind III
Restriction fragments after digest with Hind III and BamH I
4018 bp
4706 bp
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restriction analysis of pKAN-R and pARA
Prediction for restriction gel
M K+ K- A+ A-M K+ K- A+ A-
500
1000
1500
20003000400050008000
10000
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ligation of pKAN-R/pARA restriction fragments
sticky endBamH I
sticky endHind III
sticky endBamH I
sticky endHind III
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ligation of pKAN-R/pARA restriction fragments
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
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restriction analysis of pKAN-R and pARA
M K+ K- A+ A-
500
1000
1500
20003000400050008000
10000
Confirmation of restriction and ligation
L M K+ K- A+ A- L
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preparing competent cells for transformation
Lipid bilayer(inner)
Lipid bilayer(outer)
Peptidoglycanlayer
Adhesion zone
Calcium ions
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transforming Escherichia coli with pARA-R
Recombinant Plasmids
Competent Cells
pARA-R
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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 expression
araC gene rfp genePBAD
Transcription
mRNA
Translation
araC protein
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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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RFP
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purification of RFP from an overnight culture
Overnightculture
Cell pelletwith RFP
Lysedcells
Pelletcell debris
RFP withbinding buffer