Biotechnology Lab Program rpGLO Recombinant GFP Expression Sequence

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Biotechnology Lab ProgramrpGLO Recombinant
GFP Expression Sequence

-Bruce Wallace
PowerPoint by Lyn Koller
Laboratory protocols by Marty Ikkanda
Pierce College, Woodland Hills
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Restriction Analysis of pDRK and pGRNOriginal
Plasmids
ara C

H
i
n
d
I
I
I
ampr
PBAD
gfp
p
D
R
K
p
G
R
N
H
i
n
d
I
I
I
H
i
n
d
I
I
I
kanr
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Restriction Analysis of pDRK and pGRNRestriction
Fragments after digest with HindIII
kanr
ara C
PBAD
ampr
gfp
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Restriction Analysis of pDRK and
pGRNPrediction for restriction gel
M D D- G G-
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Ligation of pDRK/pGRN Restriction Fragments
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Ligation of pDRK/pGRN Restriction
FragmentsRecombinant Plasmid of Interest
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Restriction Analysis of pDRK and
pGRNConfirmation of restriction and ligation
M D D- G G- L
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Preparing Competent Cells for Transformation
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Transforming Escherichia coli With a Recombinant
Plasmid
Recombinant plasmids
transformed cells
Competent cells
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Transforming Escherichia coli With a Recombinant
Plasmid
adhesion zone
Peptidoglycan layer
phospholipids
calcium ion
plasmid
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Growth of transformed bacteria on various plates
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 GFP
ExpressionColony isolation and culture
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GFP Expression
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GFP Expression
ara C gene
gfp gene
promoter
C protein prevents gfp transcription by causing a
loop to form in the region of the gfp gene
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GFP Expression
Arabinose C protein complex prevents DNA
looping and helps to align RNA polymerase on the
promoter site.
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GFP
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Purification of GFP from an Overnight Culture