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Team:Chiba/System 1/Result
From 2010.igem.org
Version 1 :
Requirement of realizing genetic double click system
1. T7 RNAP pulse in response to 1st and 2nd input
In response to 1st and 2nd input, T7 RNAP has to express as pulse.
To accomplish this, Lux/CI434 hybrid promoter should be activated or repressed stringently.
So, we checked this part.
2. CI repression
CI needs to work as a repressor of T7/CI-OR1 hybrid promoter at 1st input and needs to
have degradated before 2nd input not to work as a repressor at 2nd input.
To accomplish this, T7/CI-OR1 hybrid promoter should be activated or repressed stringently.
So, we checked this part).
3. Setting the fixed time
There is the fixed time between 1st and 2nd input.
To accomplish this, CI has to re-express after washout. (Data not shown)
1-2. Pulse generator
Protocol
E. coli strain DH10B was used for the pulse-generator experiments. Co-transformed cell were pre-cultured in test tubes for overnight at 37C, 200rpm. Dilute the culture 1:100 into 2ml of fresh medium and grow for an additional 5 hours in Supplemented M9 medium . For the liquid experiments, expression was induced at the log phase (OD600 of 0.4) by the addition of AHL (3OC6HSL)at the appropriate concentration. One-milliliter samples were taken every 5 min.
Result
There are no obvious pulse observed.We use destabilized GFP at the downstream of pLux/cI434 hybrid promoter as a positive control, but it also didn't generate any fluorescent.Maybe because we use weak RBS and GFP with LVA, the GFP protein degradation is too soon to be observed. Our project focus on T7 RNA Polymerase pulse and the output GFP will be amplified. So we hope there will be GFP pulse generated when co-transform plasmid 1 and 2.
