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

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pARA-R construct

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

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Engineering the Plasmid: ligation of rfp gene into p-ARA

sticky endBamH I

sticky endHind III

sticky endBamH I

sticky endHind III

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Restriction digest of pARA-R

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

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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

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Restriction analysis of pARA-R

Prediction for restriction gel

M A+ A-

500

1000

1500

20003000400050008000

10000

M A+A-

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pARA-R construct

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

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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

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