4/3/14: O/N in LB 50mM MOPS pH 7.

0, +/- 1% arabinose


Summary: Double mutant should not have any increased reporter activity.



4/4/14: O/N in LB 50mM MOPS pH 7.0, +/- 5% arabinose

Summary: Changed to 5% arabinose to try to increase between +/- arabinose conditions. Now,
- arabinose reporter activity is higher across the board, which is opposite of what is expected.





0
5000
10000
15000
20000
25000
30000
35000
+ ara - ara + ara - ara + ara - ara + ara - ara + ara - ara + ara - ara + ara - ara
PAO1 sadC sadC siaD siaD sadCsiaD sadCsiaD
0
5000
10000
15000
20000
25000
+ ara - ara + ara - ara + ara - ara + ara - ara + ara - ara + ara - ara + ara - ara
PAO1 sadC sadC siaD siaD sadCsiaD sadCsiaD




4/8/14: O/N in LB 50mM MOPS pH 7.0, +/- 5% arabinose

Summary: Repeated and still see opposite effect when using 5% arabinose.






4/10/14: 3hrs in LB 50mM MOPS pH 7.0, +/- 5% arabinose

Summary: 5% arabinose seems to be interfering with PcdrA promoter activity, even at an earlier
time point.
0
5000
10000
15000
20000
25000
30000
+ ara - ara + ara - ara + ara - ara + ara - ara + ara - ara + ara - ara + ara - ara
PAO1 sadC sadC siaD siaD sadCsiaD sadCsiaD
0
5000
10000
15000
20000
25000
30000
35000
+ ara - ara + ara - ara + ara - ara + ara - ara + ara - ara + ara - ara + ara - ara
PAO1 sadC sadC siaD siaD sadCsiaD sadCsiaD
4/11/14: wt PAO1 P
BAD
-psl P
cdrA
::lacZ grown to late-log in LB 50mM MOPS pH 7.0, +/- 5%
arabinose for less than 1 minute before lysing cells and starting assay. Three bars are technical
replicates of lysis step.

Summary: Arabinose molecule itself does not interfere with B-gal assay.





4/14/14: All strains with lower amounts of arabinose, 3 hours. Cells lysed and frozen O/N.
Summary: 0.5% arabinose is best for assay. SadC clone 8 always has the best in this assay. Re-
sequence others? Also, double mutant has weak effect of added arabinose when it should have
zero effect.

0
10000
20000
30000
40000
50000
60000
+ ara - ara
PAO1
0
20
40
60
80
100
120
140
160
n
o

a
r
a
0
.
5
%
1
%
n
o

a
r
a
0
.
5
%
1
%
n
o

a
r
a
0
.
5
%
1
%
n
o

a
r
a
0
.
5
%
1
%
n
o

a
r
a
0
.
5
%
1
%
n
o

a
r
a
0
.
5
%
1
%
n
o

a
r
a
0
.
5
%
1
%
wt PAO1 sadC 7 sadC 8 siaD 2 siaD 6 sadCsiaD 6 sadCsiaD 8
4/14/14: All strains in LB vs. 24 hour PAO1 cell-free Sup, 6 hours. Cells lysed and frozen O/N.
Should do Psl blot on frozen Sup (-20C) to semi-quantify Psl.




4/15/14: Experiment from 4/14 did not repeat. Strains with arabinose have less activity again



Summary:
5% arabinose is too much for the assay0.5% is best.
PcdrA promoter is inactivated at very high levels of Psl transcription / extracellular Psl?
4 hour Pa Sup seems to have same inhibitory effect as 5% arabinose didpossibly due to
high level of Psl in sample?
Having trouble efficiently lysing cells (especially with Psl overproduced)? Vortex longer
/ use lowed cell density in assay.
Put PcdrA::gfp plasmid into reporter strains as a second readout of promoter activity +/1
arabinose.
0
20
40
60
80
100
120
140
160
LB Sup LB Sup LB Sup LB Sup LB Sup LB Sup LB Sup
wt sadC 7 sadC 8 siaD 2 siaD 6 sadCsiaD 6 sadCsiaD 8
0
10000
20000
30000
40000
50000
60000
L
B
0
.
5
%
1
%
L
B
0
.
5
%
1
%
L
B
0
.
5
%
1
%
L
B
0
.
5
%
1
%
L
B
0
.
5
%
1
%
L
B
0
.
5
%
1
%
L
B
0
.
5
%
1
%
wt sadC 7 sadC 8 siaD 2 siaD 6 sadCsiaD 6 sadCsiaD 8