Download Group 4 Data - Mike DeSalvio

Group 4 Data
Diane Meas
The 3 A-Michaels (get it??)
3 amigos… a-michaels….
A. baumannii def
• Gene name: Zinc (II) binding peptide deformylase 1
• Sequence:
ATGGCCTTATTACCTATTTTAAGTTTTCCTGATCCCCGTCTTCGTACCATTGCTAAGCCC
GTTGAAGAAGTTACTGATGAAATTCGTCAACTTGCAGCAGATATGTTTGAAACCATGT
ATGCGGCACCAGGTATCGGTTTAGCAGCTTCTCAGGTCGATCGTCATATTCAGCTTATC
GTCATGGATTTGTCTGAATCTAAAGATGAACCTATGGTTTTCATTAACCCCAAAGTAAC
TCCGCTGACTGAAGAGACGCAGCCGTACGAAGAAGGCTGTCTATCAGTGCCACAAAT
ATACGACAAAGTTGATCGCCCGTCACGCGTGAAAATTGAGGCAATTAACCTTGAAGG
TCAAGCATTTGAGATAGAAGCTGACGGACTTCTCGCTGTTTGTATCCAACATGAAATG
GATCACTTAAATGGCAAATTGTTTGTGGATTATTTGTCGCCACTTAAGCGTCAGCGTG
CGCGTGAAAAAGTCGAAAAAATTGTTCGCCAACGCGAACGTGAAAAAGTTGCGGTA
AAACGTTAA
A. Baumannii def
• Amino Acid Sequence
• MALLPILSFPDPRLRTIAKPVEEVTDEIRQLAADMF
ETMYAAPGIGLAASQVDRHIQLIVMDLSESKDEPM
VFINPKVTPLTEETQPYEEGCLSVPQIYDKVDRPSRV
KIEAINLEGQAFEIEADGLLAVCIQHEMDHLNGKLF
VDYLSPLKRQRAREKVEKIVRQREREKVAVKR
Primer Design
• Forward Primer:
–
–
–
–
5’-GACGACGACAAGATGGCCTTATTACCTATTTTAAGTTTTCCT-3’
Tm = 59°C
Primer name: Gp4Abdef_F
In red: Forward LIC tail
• Reverse Primer:
–
–
–
–
5’-GAGGAGAAGCCCGGTTAACGTTTTACCGCAACTTTTTC-3’
Tm = 58°C
Primer name: Gp4Abdef_R
In blue: Reverse LIC tail
PCR: A. baumannii def gene
1 2 3 4 5 6
(a)
7
8
(b)
5000
1500
500
Figure 1. (a) PCR of Acinetobacter baumannii def gene using LIC primers. The expected
product size is 561 bp. Lane 1: A. baumannii A1; Lane 2: A. baumannii A2; Lane 4:
GeneRuler™ 1kb DNA Ladder; Lane 5: A. baumannii B1; Lane 6: A. baumannii B2;
Lane 8: 100 kb ladder; Lanes 3 and 7 are empty. (b) GeneRuler™ 1kb DNA Ladder
band sizes.
Restriction Digest with BglII and BamHI
1
2
3
4
5000
1500
500
Figure 2. Double restriction digest of A. baumannii def PCR products using BglII and
BamHI. The expected results were two bands at 561 bp and 5000 bp corresponding
to the insert and vector sizes respectively. Our results show one band at
approximately 6 kb for Lanes 2 and 3. Lane 1 & 4: GeneRuler™ 1kb DNA Ladder;
Lane 2: Ab Def Clone 1 (~6 kb); Lane 3: Ab Def Clone 2 (~6kb).
Restriction Digest with BglII and BamHI for Ab
Def 3 to 8
1
2
3 4
5
6 7
5000
1500
500
8
Figure 3. Double restriction digest
with BglII and BamHI for Ab Def
clones 3 to 8. Lanes 1 and 5:
GeneRuler™ 1kb DNA Ladder.
Lane 2: Ab Def Clone 3 (~6kb);
Lane 3: Ab Def Clone 4 (~6kb);
Lane 4: Ab Def Clone 5 (~6kb);
Lane 6: Ab Def Clone 6 (~6kb);
Lane 7: Ab Def Clone 7 (~6kb);
Lane 8: Ab Def Clone 8 (~6kb).
Restriction Digest with HincII
1
2
3 4
5 6
5000
1500
500
Figure 4. Acinetobacter baumannii
def clones 1 and two were digested
using the restriction endonuclease
HincII. We expect 3 bands at
approximately 3921, 1556, and ~500
bp. After visualization with
ethidium bromide staining, we see a
bands at ~6000 bp, 4000 bp, 1500
bp for Ab def clone 1. For Ab def 2,
we see bands at ~6000 bp, 4000 bp,
15000 bp, and a very faint band at
500 bp. Lanes 1 and 6: GeneRuler™
1kb DNA ; Lane 2: Ab Def clone 1;
Lane 5: Ab Def clone 2; lanes 3 and
4 are empty.
Group 4: Restriction Digest of TR-45, 46, 49, 50, 53, 54, 57, 58
with BamHI, and Double Digests of Ab Def 1, 2, 3, and 4 with
BglII and BamHI
1
2
3 4
5
6 7
8
~4kb
~1.2kb
9 10 11 12 13 14 15 16
~4kb
~1.2kb
Figure 5. Restriction digests of TR-45, 46, 49, 50,
53, 54, 57, and 58 with BamHI and double digests
of Ab Def 1, 2, 3, and 4 with BglII and BamHI. The
TR clones all show two bands at approximately 4kb
and 1.2 kb. All of the Ab Def clones double digests
show a single band at approximately 5.5kb. The Ab
def clones do not appear to be cut by the two
enzymes. Lanes 1, 8, 9, 16: GeneRuler™ 1kb DNA
Ladder; Lane 2: TR-45; Lane 3: TR-46; Lane 4: TR49; Lane 5: TR-50; Lane 6: TR-53; Lane 7: TR-54;
Lane 10: TR-57; Lane 11: TR-58; Lane 12: Ab Def 1
Double Digest (DD) #2; Lane 13: Ab Def 2 DD #2;
Lane 14: Ab Def 3 DD #2; and Lane 15: Ab Def 4 DD
#2 .
Group 4: Restriction Digest of TR-45 to TR-55 with BamHI
1
2
3
4
5
6
7
8
9
10 11 12
5000
1500
500
Figure 6. Restriction digest of TR-45 to TR-55 clones with
BamHI. All TR clones show a two bands at approximately
4kb and 1.2 kb. Lane 1: GeneRuler™ 1kb DNA Ladder;
Lane 2: TR-45; Lane 3: TR-46; Lane 4: TR-47; Lane 5: TR48; Lane 6: TR-49; Lane 7: TR-50; Lane 8: TR-51; Lane 9:
TR-52; Lane 10: TR-53; Lane 11: TR-54; and Lane 12: TR55.
Group 4: Restriction Digest of TR-56 to TR-65 with BamHI and
Double Digest of Ab Def 2 with BglII and BamHI
1
2
3
4
5
6
7
8
9
10 11 12
5000
1500
500
Figure 7. Restriction digest of TR-56 to TR-65 with
BamHI and double digest of Ab Def clone 2
(overnight) with BglII and BamHI. The TR clones all
have two bands at approximately 4kb and 1.2 kb. Ab
def clone 2 shows a band at approximately 5kb and
500 bp. Lane 1: TR-56; Lane 2: TR-57; Lane 3: TR-58;
Lane 4: GeneRuler™ 1kb DNA Ladder; Lane 5: TR-59;
Lane 6: TR-60; Lane 7: TR-61; Lane 8: TR-62; Lane 9:
TR-63; Lane 10: TR-64; Lane 11: TR-65; and Lane 12:
Ab Def 2 Double Digest #3 with BglII and BamHI.
Group 4: SDS gel of His-tag Protein Overexpression and
Purification of AbFabI and AbDef
(a)
kDa
225
50
35
25
10
1
2 3
4
5 6
(b)
Figure 8. SDS gel of His-tag protein
overexpression and purification of AbFabI and
AbDef. For AbFabI we expect a band at ~35.5
kDa and for AbDef we expect a band at ~24.6
kDa.
(a)Lanes 1 and 6: Perfect Protein™ Markers
10-225 kDa; Lane 2: Group 4 AbDef (5 hour
induction) 10 µl sample + 10 µl 2x SDS
loading dye; Lane 3: group 4 AbDef (5 hour
induction) 20 µl sample + 20 µl 2x SDS
loading dye; Lane 4: AbFabI (overnight
induction) 10 µl sample + 10 µl 2x SDS
loading dye; and Lane 5: AbFabI (overnight
induction) 20 µl sample + 20 µl 2x SDS
loading dye.
(b)Perfect Protein™ Markers 10-225 kDa
protein size determination.
Group 4: SDS gel of EcFabI overexpressed
His-tag protein (large scale 250 ml culture)
1
2
kDa
225
50
35
25
10
Figure 9. His-tagged EcFabI protein overexpression (large scale)
SDS gel. We expect an overexpressed band at approximately 32.3
kDa.
Lane 1: EcFabI (15 µl protein extract + 15 µl 2x SDS loading dye);
Lane 2: 5 µl of Perfect Protein™ Markers 10-225 kDa.
Group 4: SDS gel of His-tagged protein overexpression of AbFabI
crude protein extract and EcFabI affinity column purification
1
2 3 4
5
6
7 8
kDa
225
50
35
25
10
Figure 10. SDS gel of AbFabI crude protein extract and
EcFabI flow through, wash, and elute. Expected protein
bands for AbFabI is ~35.5 kDa and for EcFabI is ~32.3 kDa.
Lane 1: AbFabI crude extract (15 µl sample + 15 µl 2x SDS
loading dye);
Lane 2: empty;
Lane 3: Perfect Protein™ Markers 10-225 kDa;
Lane 4: EcFabI flow through 1;
Lane 5: Ec FabI wash 1;
Lane 6: EcFabI Elute 1;
Lane 7: empty;
Lane 8: Perfect Protein™ Markers 10-225 kDa.
SDS gel of His-tagged protein overexpression of E. coli and A.
baumannii FabI “Elute” Fraction
1
2
3
4
5
kDa
225
50
35
25
10
6
7
Figure 11. SDS PAGE gel of EC FabI (large
scale) and AB FabI Elute. Expected band for
EC FabI is ~32.3 kDa while AB FabI is ~35.5
kDa.
Lane 1: Ec FabI Elute 2.
Lane 2: Ec FabI Elute 3.
Lane 3: Ec FabI Strip.
Lanes 4 and 5: Empty.
Lane 6: Perfect Protein™ Markers 10-225 kDa.
Lane 7: Ab FabI Elute 1.
Lanes 1 to 3 are from the 250 ml culture scale
up. Lanes 1 to 3 shows a distinct band at ~35
kDa, which will correlate with our gene. It
also contains at ~75 kDa of unknown origin.
There was no significant over expression in
the AB FabI gene (Lane 7)
SDS-PAGE of EcFabI small scale
protein extraction and purification
kDa
225
50
35
25
1
2
3 4
5 6
Figure 12. SDS-PAGE of small scale E. coli fabI
protein extraction and purification.
Lane 1: Perfect Protein™ Markers 10-225 kDa;
Lane 2: EcFabI crude extract;
Lane 3: EcFabI flow through fraction;
Lane 4: EcFabI Wash fraction;
Lane 5: Ec FabI Elute;
Lane 6: EcFabI Strip fraction