rpARA Recombinant mFP Expression Sequence

1
biotechnology lab program
pARA-R Recombinant
RFP Expression Sequence
Laboratory Protocols by
Marty Ikkanda
Powerpoint by
Anthony Daulo
Pierce College, Woodland Hills, CA
V.1.2.2
2
restriction analysis of pKAN-R and pARA
pKAN-R
5408 bp
BamH I
rfp 702 bp
pARA
4058 bp
Hind III
BamH I
Hind III
40 bp
3
restriction analysis of pKAN-R and pARA
Restriction fragments after digest with Hind III
and BamH I
BamH I
Hind III
4018 bp
BamH I
Hind III
4706 bp
BamH I
Hind III
702bp
BamH I
Hind III
40 bp
4
restriction analysis of pKAN-R and pARA
Prediction for restriction gel
M
K
K-
A
A-
5
ligation of pKAN-R/pARA restriction fragments
3
5
3
5
5
3
5
5
3
3
3
5
6
ligation of pKAN-R/pARA restriction fragments
Recombinant plasmid of interest
pARA-R
4720 bp
BamH I
rfp 702bp
Hind III
7
restriction analysis of pKAN-R and pARA
Confirmation of restriction and ligation
M
K
K-
A
A-
L
8
preparing competent cells for transformation
Lipid bilayer (inner)
Peptidoglycan layer
Adhesion zone
Lipid bilayer (outer)
9
transforming Escherichia coli with pARA-R
Recombinant Plasmids
10
transforming Escherichia coli with pARA-R
Lipid bilayer (inner)
Peptidoglycan layer
Adhesion zone
Lipid bilayer (outer)
11
growth of transformed bacteria on various plates
P plates
LB
LB/amp
LB/amp/ara
P- plates
No growth
LB
LB/amp
12
preparing an overnight culture of E. Coli for RFP
expression
Colony isolation and culture
13
RFP expression
Transcription
Translation
14
RFP expression
araC protein
15
RFP expression
Arabinose araC protein complex prevents DNA
looping
and helps to align RNA polymerase on the promoter
site (PBAD).
arabinose
Translation
RNA polymerase
araC protein
arabinose araC protein complex
16
RFP
17
RFP
18
purification of RFP from an overnight culture
Overnight culture
Cell pellet with RFP
Lysed cells
Pellet cell debris
RFP with binding buffer