<div class="p-size">Overnight culturing Ecoli BL21-4A5-R-IG and determinating its fluorescence, we found that the fluorescence is low( Table 1). The system cannot be opened through its background expression, reflecting the positive feedback system is weak. It didn’t match with our expection. We speculated that phenomenon appeared because of the LuxI protein’s low effective expression, could not produce a lot of AHL. Therefore verified module LuxI. The fluorescence values were significantly increased by adding different concentrations of AHL(Table 2) . The system was opened which indicating there was a problem with the LuxI module. Analysis of LuxI gene sequence, we found out the LuxI cloned down from iGEM part() has LVA tags.The tag will speed up the degradation rate of the corresponding protein, making the LuxI protein in cell can not play a stable role.The AHL signal factor was too weak to open the positive feedback system.</br></div>

<div class="p-size">Fragmentating of Ecoli BL21-4A5-R-IG- △ LVA and additing AHL at the same time,then measuring fluorescence values compared with control which was without AHL. The relative fluorescence value of test group increased significantly(Figure 3). We could see the system is open, and the positive feedback effect is normal. After 20 hours, it was found that the AHL-free control group was also up to xx, that is, the system background expression made the system open and the positive feedback system was constructed successfully.</br></div>

<div class="p-size">In order to avoid the impact of system detection limits or detection time

After addition of IPTG inoculation, the results showed that the positive feedback effect was normal; without adding IPTG, the positive feedback effect was closed (Table 3), indicating that the plux / Lac switch can work properly.</br>

In the stationary phase of cell growth, IPTG induction was added. After 12h induction, no positive feedback was found, the system did not work normally,either. (Figure 4)</br>

At smooth period,different concentrations of AHL were added in medium, positive feedback system still did not work (Figure 5). And then we accessing to a large number of literature to explore the reasons .Ultimately ,we speculated that LuxR may be in the stable period of E. coli did not work and led to positive feedback system failure (reference  1). （If you want to read more details, you can click here to see our contribution）</br>

So we would rather choose logarithmic growth period than stationary phase to detect the input signal.</br></div>

<div class="p-size">Relative fluorescence of Ecoli BL21-4A5-RG and Ecoli BL21-4A5-R-IG- △ LVA were measured after added different concentrations AHL. Ecoli BL21-4A5-R-IG- △ LVA measurement accuracy was 10-9M while Ecoli BL21-4A5-RG was 10-8M (Figure 7). As for the ordinary high performance liquid chromatography,when the concentration reached 10-6M (Figure 8), Its detection was not very obvious. Ecoli BL21-4A5-R-IG- △ LVA compared with the Ecoli BL21-4A5-RG, the measurement accuracy was higher than an order of magnitude; compared with high performance liquid chromatography, the measurement accuracy was higher than three orders of magnitude. To achieve the accuracy of 10-9M, you can also use the method of chromatography mass spectrometry, but its operation method will be a little complex. (reference  2)</br></div>

<div class="p-size">A higher background expression can affect the detection limits of the system. AiiA hydrolytic enzymes can hydrolyze the presence of AHL and LVA tag labels to accelerate the degradation of LuxI, thereby reducing the generation of AHL . Both are able to  control AHL background expression . However, the effect of LVA tag on AHL background expression will increase with the enlargement of positive feedback system. The reason is that the tag exists on LuxI. With the expression of LuxI, the total number of intracellular tags will increase accordingly. Leading to its inability to turn on the positive feedback effect (1 result) before the stationary period. While the AiiA hydrolase is different. According to the Michaelis-Menten equation, when the AHL is enough, the degradation rate of AHL is constant, so using AiiA to control background expression will not have a significant effect on the positive feedback effect. Considering, we chose to add AiiA for system control. Compared with Ecoli BL21-4A5-R-IG- △ LVA, the addition of AiiA will delay the threshold, but the  increase  rate is basically same as before(Figure 9), in line with our expectations. The decrease in the end point may because  the positive feedback effect had already over after cell reached the stationary period .It also confirmed our previous speculation that LuxR did not work at stationary period.</br></div>

<div class="p-size">Isolation Ecoli BL21-4A5-AiiA/1C3-IT containing both pSB4A5-R-IG- △ LVA-AiiA and pSB1C3-LuxI-TetR plasmids, added into different concentrations of tetracycline. The results showed that there was no significant difference between different concentrations Threshold, the system detection performance of Tetracycline is not high.</br>

Construction of Ecoli BL21-1C3-RT to detect whether the converter is normal or not.Finding out the background level was high, meaning the double plasmid system needed more AiiA hydrolase to control background expression of AHL.Initially, we intended to switch to a stronger promoter or RBS to improve this system, but because we have little time, the improvment hasn’t been done.</br>