1. Synthetic Circuit - I : pSB3C5/Pars-gfp-Pznt-rfp
1)Pznt-rfp
Zn2+ (mM)
0
0.5
0.75
1
1.5
2
OD600
0.490 ± 0.003
0.459 ± 0.004
0.433 ± 0.008
0.418 ± 0.005
0.275 ± 0.011
0.243 ± 0.008
Red fluorescence (RF)
26628 ± 706
27856 ± 292
29333 ± 292
31217 ± 66
46044 ± 237
49188 ± 174
OD600/RF
54376 ± 1203
60646 ± 787
67803 ± 573
74687 ± 755
167833 ± 7202
202565 ± 6428
Background
0
6270 ± 520
13427 ± 1719
20311 ± 475
113457 ± 6016
148189 ± 6731
This parts is producing red fluorosence based on the zinc ion level. The zinc detecting promoter can be
induced beyond 1mM concentration of zinc ion. This range from 1mM to 2mM is proper to detect zinc level on
the common waste water. Maybe it is required to detect the upper concentration of zinc ion in waste water that
using the dilution method which dilute the level of zinc concentration. If the level curve is too rugh or not
too much change according to concentraion, you need to apply dilution or concentration of sample.
2)Pars-gfp
Differing from zinc ion, Arsenic ion is more severe impact on the E.coli. When microbials are exposed by
arsenic ion even 1mM concentration, This concentraion of arsenic ion is already full of toxicity. It is very
senstive to detect arsenic ion. It means that arsenic ion can provide strong harmful effect on the organism
even microbial which is known as the best survivor on the earth. The interaction of arsenic ion is participated
in more than two or more group of proteins or others. See the curve of fluorosence of GFP.the level curve is
show sigmoid curve form.
It is supose that the the cost of the expression of arsenic ion binder is very expensive for microbial.
Because even lower level of concentraion the fluorosence curve did not show regression curve at the initiation
point. Strong inhibitor can be exist at the initation of expression state.
The main point of this idea is that the possible measure range. Although the view of whole curve show sigmoid
curve, the range from 0.15 to 0.5 mM concentration of arsenic ion show regression. We can utilize this midpoint
of curve to measuring arsenic ion rather than endpoint of this curve. Also we can use the dilution method if the sample contain too much concentration of arsenic ion.
We belive that the hypothesis can be accepted we just explain above. Because the population size of E.coli
could determine the intensity of fluorosence. Comparing to zinc ion, the arsenic ion is more harmful effect on
the growth of bacteria. This point is the very critical delema in terms of that the bacteria can be killed by
the measuring molecules. It is hard to distinguish whether the effect of concentration of sample or death rate
of bacteria.
AsO3- (mM)
0
0.00625
0.125
0.25
0.5
0.75
1
OD600
0.553 ± 0.009
0.519 ± 0.002
0.480 ± 0.005
0.395 ± 0.007
0.315 ± 0.006
0.286 ± 0.004
0.273 ± 0.006
Green fluorescence (GF)
27740 ± 226
26411 ± 169
25267 ± 267
25536 ± 213
25407 ± 259
24849 ± 152
26479 ± 149
OD600/GF
50206 ± 1225
50855 ± 123
52606 ± 689
64669 ± 1684
80665 ± 603
86897 ± 1321
96784 ± 2165
Background
0
648 ± 1314
2399 ± 969
14463 ± 2896
30459 ± 1631
36691 ± 1636
46578 ± 3220
2.Synthetic circuit – II : PyodA-AMD
Cd2+ (mM)
0
0.05
0.1
0.2
0.4
0.8
OD615
0.242 ± 0.005
0.258 ± 0.008
0.274 ± 0.011
0.284 ± 0.01
0.293 ± 0.011
0.302 ± 0.01
Background
0
0.016 ± 0.004
0.032 ± 0.006
0.043 ± 0.005
0.052 ± 0.007
0.060 ± 0.007
Cadimum detector has similar problem related to the toxicity issue just above result. Also cadimum ion has
harmful effect on the even bacteria. 0.2mM of concentration of cadimum is enough to inhibt the growth of
E.coli. The E.coli did not show big difference at between 0.2mM and 0.4mM of concentration of cadimum.
In addition,we try to show the change according to the concentration of cadimum ion in the sample with naked
eye. Amd gene which can convert form acetoaminpen to brwon color only demonstrate the possiblity to those
hypothesis. Although we know the more strong promoter to interact heavy metals, we do not use this due to
standards issue. Some parts from MIT did not work even not accurate sequenced.
GFP and RFP fluorsence can see only with UV irritation.