Cell Surface Targeting cont

1
Cell Surface Targeting contd

• 7/17/06

2
Adaptamers
3
Update

• Initial PAGE experiment did not work protein was
  run off gel.
• Following some testing (collaboration with Nano
  group!) we got a gel out.

4
Gel
4Thromb T35
5Thromb T50
2Thromb T5
3Thromb T20
6Strep
10Strep S50
Thromb
.1 BSA
12 Lane 4 9
7Strep S5
9Strep S35
8Strep S20
11 Lane 3 8
1Thromb
T5
T20
T35
T50
Strep
S5
S20
S35
S50
5
4Thromb T35
5Thromb T50
2Thromb T5
3Thromb T20
10Strep S50
6Strep
Thromb
BSA
12 Lane 4 9
7Strep S5
9Strep S35
8Strep S20
11 Lane 3 8
1Thromb
T5
T20
T35
T50
Strep
S5
S20
S35
S50
6
Effect of BSA?

• Lane 1 .1 BSA
• Lane 2-5 Thrombin in .1 BSA

65 kDa thrombin
53 kDa streptavidin
Thrombin subunit

• Why is subunit band so dark?

7
4Thromb T35
5Thromb T50
2Thromb T5
3Thromb T20
10Strep S50
6Strep
Thromb
BSA
12 Lane 4 9
7Strep S5
9Strep S35
8Strep S20
11 Lane 3 8
1Thromb
T5
T20
T35
T50
Strep
S5
S20
S35
S50
8
Lack of conclusion

• No evidence of aptamer binding
• Problems?
• pH, voltage, staining
• Next
• Visualize where DNA is
• Assay for streptavidin binding to biotin

9
In other news

• LPS aptamer group got back to us.
• LPS aptamers developed for a phase I SBIR (Small
  Business Innovation Research) funding was cut,
  so they dont have a sequence.

10
BioBricking streptavidin
11
BioBricking streptavidin
Clones from Ting lab StrepW (wild-type) StrepH
(His6 tag) StrepD (dead)
12
BioBricking streptavidin
Clones from Ting lab StrepW (wild-type) StrepH
(His6 tag) StrepD (dead)
StrepF
StrepMF
E X
390
1
S P
StrepMR
StrepR
XbaI site, 120
TCTAGA AGCCGC
13
BioBricking streptavidin
Clones from Ting lab StrepW (wild-type) StrepH
(His6 tag) StrepD (dead)
StrepF
StrepMF
E X
390
1
S P
StrepMR
StrepR
XbaI site, 120
TCTAGA AGCCGC
No mutagenesis
StrepH
StrepW
StrepD
400
300
F/R
200
100
14
BioBricking streptavidin
Clones from Ting lab StrepW (wild-type) StrepH
(His6 tag) StrepD (dead)
StrepF
StrepMF
E X
390
1
S P
StrepMR
StrepR
XbaI site, 120
TCTAGA AGCCGC
No mutagenesis
Mutagenesis
StrepW
StrepH
StrepD
StrepH
StrepW
400
StrepD
400
300
F/R
300
200
F/R
R/MF
200
100
F/MR
100
15
Digest w/ XbaI and PstI
16
Digest w/ XbaI and PstI
17
Digest w/ XbaI and PstI
1
444
295
296
Streptavidin BioBrick
18
Digest w/ XbaI and PstI
E X(23)
S P(430)
(XbaI) 146
1
444
295
296
Streptavidin BioBrick
1495 PstI
19
Digest w/ XbaI and PstI
E X(23)
S P(430)
(XbaI) 146
1
444
295
296
Streptavidin BioBrick
305 PstI
345 XbaI
1495 PstI
20
Digest w/ XbaI and PstI
E X(23)
S P(430)
(XbaI) 146
1
444
295
296
Streptavidin BioBrick
305 PstI
345 XbaI
StrepW
StrepH
StrepD
3000
2000
1650
1000
850
650
500
400
300
200
1495 PstI
100
non-mut
mut
21
Biobricking full-length OmpA
22
Biobricking full-length OmpA
Cloned from K12 genome No mutagenesis needed
23
Biobricking full-length OmpA
Cloned from K12 genome No mutagenesis needed
E X
OmpAF
1041
1
OmpAR
S P
24
Biobricking full-length OmpA
Cloned from K12 genome No mutagenesis needed
E X
OmpAF
1041
1
OmpAR
S P
1st PCR (100ng genomic DNA)
1000
25
Biobricking full-length OmpA
Cloned from K12 genome No mutagenesis needed
E X
OmpAF
1041
1
OmpAR
S P
2nd PCR
1st PCR (100ng genomic DNA)
1000
1000
26
Progress/Plans

• BioBrickd streptavidin clones from Ting lab
  (StrepW, H, D)
• BioBrickd full OmpA
• Recd single-chain dimeric streptavidin clones
  from Filiz Aslan (SCD-NM, C2, E2) transformed
  and miniprepped
• Send StrepW, StrepH, StrepD, and full OmpA clones
  for sequencing
• BioBrick PCR
• OmpA(46-159) and (46-66)
• Lpp segment from K12 genome
• SCD-NM, C2, E2 (mutagenesis needed for PstI site)
• Assembly?