Part:BBa_K3805138:Experience

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Experiments of BBa_K3805138

For the synthesis of AIP by agrB-D and the verification of toggle switch functionality using AIP, we also carried out the related experiments

Experiment1：Toggle Switch Verification

To verify the Bi-stable Switch, we replaced our aimed protein gene with GFP sequence. When a certain amount of exogenous AIP was applied to the bacterial fluid, red fluorescence(mCherry) was observed under the microscope. We then applied a concentration gradient of synthetic AIP to the bacterial fluid to establish an accurate coordination between AIP concentration and mCherry secretion and plotted the corresponding curve.

Experiment 2: Measurement of AIP generation

stage1:Measurement of standard experimental data

We incubated the guards overnight for 12-16h, and then split the experimental material in the ultra-clean bench: five wells of a 96-well plate were poured with 200ul of bacterial solution, followed by 1mM of AIP, and 1ul each of diluted 107, 108 and 109 AIP into the first four wells, leaving a control of the original bacterial solution without AIP.

Then we put the partitioned experimental material into the enzyme marker to detect the fluorescence intensity of mcherry and record the experimental data

stage 2: Deceiver data collection After collecting the standard experimental data, we filtered the 12h culture of the deceiver and mixed the remaining culture with an equal amount of the guard's bacterial solution. 200ul of the culture was placed in a 96-well plate and the mcherry was measured under the ELISA.

result

experiment1

When a certain amount of exogenous AIP was applied to the bacterial fluid, red fluorescence(mCherry) was observed under the microscope. After a period of time, red fluorescence disappeared, and green fluorescence was observed under the microscope.

experiment2

After a period of testing, we finally got our experimental results

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