para-r sequence rfp expression sequence biotechnology lab program laboratory protocols by: marty...
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![Page 1: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/1.jpg)
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
![Page 2: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/2.jpg)
pARA-R construct
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
Bruce Wallace
![Page 3: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/3.jpg)
Engineering the Plasmid: ligation of rfp gene into p-ARA
sticky endBamH I
sticky endHind III
sticky endBamH I
sticky endHind III
Bruce Wallace
![Page 4: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/4.jpg)
Restriction digest of pARA-R
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
Bruce Wallace
![Page 5: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/5.jpg)
Restriction analysis of pARA-R
BamH I Hind III
702bp
BamH IHind III
Restriction fragments after digest with Hind III and BamH I
4018 bp
Bruce Wallace
![Page 6: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/6.jpg)
Restriction analysis of pARA-R
Prediction for restriction gel
M A+ A-
500
1000
1500
20003000400050008000
10000
M A+A-
Bruce Wallace
![Page 7: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/7.jpg)
pARA-R construct
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
Bruce Wallace
![Page 8: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/8.jpg)
Preparing competent cells for transformation
Lipid bilayer(inner)
Lipid bilayer(outer)
Peptidoglycanlayer
Adhesion zone
Calcium ions
Bruce Wallace
![Page 9: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/9.jpg)
Transforming Escherichia coli with pARA-R
Recombinant Plasmids
Competent Cells
pARA-R
Bruce Wallace
![Page 10: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/10.jpg)
Calcium ions
pARA-R
Transforming Escherichia coli with pARA-R
Adhesion zone
Lipid bilayer(inner)
Lipid bilayer(outer)
Peptidoglycanlayer
Bruce Wallace
![Page 11: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/11.jpg)
Growth of transformed bacteria on various plates
P+ plates
P- plates
LB LB/amp LB/amp/ara
LB LB/amp
No growth
Bruce Wallace
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Colony isolation and culture
Preparing an overnight culture of E. Coli for RFP expression
LB/amp/arabroth
Bruce Wallace
![Page 13: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/13.jpg)
RFP expression
araC gene rfp genePBAD
Transcription
mRNA
Translation
araC protein
Bruce Wallace
![Page 14: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/14.jpg)
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
Bruce Wallace
![Page 15: PARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce](https://reader030.vdocuments.site/reader030/viewer/2022032722/56649ce35503460f949af541/html5/thumbnails/15.jpg)
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)
Bruce Wallace
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RFP
Bruce Wallace
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Purification of RFP from an overnight culture
Overnightculture
Cell pelletwith RFP
Lysedcells
Pelletcell debris
RFP withbinding buffer
Bruce Wallace
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