x********u
发帖数: 430 | 1 I have constructed several gene circuits and got some unpredictable data (
Please open the link for the attached figure)
P: inducible promoter
P': constitutive promoter
X1 and X2: protein binding site (which will affect the expression level of
eGFP when the regulator protein binds them)
R1 and R2: putative regulator protein for sequence X1 and X2, respectively
eGFP: enhanced green fluorescence protein/reporter gene
A broad range of inducer concentration which known to bind effector R1 and
abolish its binding with X1 has been tested in this study, as denoted by
different legends in the figure.
All the four constructs reside in the same host cell with idential genetic
background.
R2 comes from another organism which known to respond to a universial
signaling molecule. After binding with this signaling molecule, R2 will
dissociate from X2.
Can anyone come up with a model to interpret this data or give some clue how
to analyze this data? My boss is very openminded and we would be pleased to
offer you proper credits if you can help us. :) we can have your name on
our paper if accepted.
Thanks. | h**********r
发帖数: 671 | 2 I have several questions about the experiment.
1). In the figures, do you use the same inducer?
2). The promoter, P has the similar function in the regulation system, X1-R1
and X2-R2?
3). R1 and R2 recognize the same inducers?
Thank you so much! | x********u
发帖数: 430 | 3 Thanks for your reply.
We use same inducer for all the experiments.
P should have same function with respect to X1-R2 and X2-R2. Once bound with
X1 or X2, both R1 and R2 should block the transcription of eGFP.
R1 and R2 recognize different inducer. The inducer for R2 is a universial
signaling molecule within all living organism.
One model currently I came up with is that X2 will interact with X1 and
decrease the binding affinity between R1 and X1, thus we saw an activation
effect when X1 is combined with X2 in Figure 2.
R2 will compete with R1 for binding with X1. At low concentration of inducer
, R1 will preferentially bind with X1 since R1 is constitutively expressed
and R2 is only minorly expressed. At high concentration of inducer (>20 from
figure 3 and 4), R2 is excessively expressed and dominate the binding with
X1.
All the cell is cultivated at exponential pahse, the intracellular pool for
this signaling molecule should be stable. When this signaling molecule is
sufficient, R2 will be saturated (with this signaling molecule) -- we saw a
first order reaction (inducer <=10). When this signaling molecule is limited
, R2 (along with R1) will interact with X1 so we saw a mixed order reaction
(inducer >=20).
A different strain with elevated level of this sinaling molecule will be
tested. The promoter activity should be shifted if we plot the promoter
activity versus inducer conc. We hope to screen strains with elevated
intracellular signaling molecule based on this results.
R1
【在 h**********r 的大作中提到】
: I have several questions about the experiment.
: 1). In the figures, do you use the same inducer?
: 2). The promoter, P has the similar function in the regulation system, X1-R1
: and X2-R2?
: 3). R1 and R2 recognize the same inducers?
: Thank you so much!
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