In order to test whether the operons we designed can perform the expected results, we first performed a computer simulation of the protein structure to preliminarily verify the function of the operon. Then,the operon is synthesized into the plasmid backbone by endonucleases and ligases.Next,the plasmid was introduced into TOP 10 competent cells, and the operon was tested for the specific function by detecting the fluorescence of E. coli.Finally,we quantitatively add the inducer to the strain culture solution to quantitatively measure the effect of the inducer concentration on the expression of transcriptional regulatory factors.

Computer simulation

Look up data in genebank
Design our Parts
Simulate the function and structure of the target protein by using amber software

Synthesis of plasmid

Entrusted company to synthesize plasmid backbone and destination sequence
Cleavage of the sequence and plasmid backbone with restriction endonucleases named BglⅡ and XhoⅠ
Use DNA ligase to join the sequence to the plasmid backbone to synthesize complete Parts . The arabinose operon on the vector plasmid is located upstream of the operon of interest, thereby conferring viability on the operon of interest. At the same time, the AMP resistance gene on the vector plasmid facilitates subsequent screening of the target colony

Preliminarily verify the function of the operon

Principle : Both the arabinose operon and our target operon produce a repressor protein, and the repressor protein binds to the operon.[1] When RNA polymerase is transcribed, it is blocked by the repressor protein, and transcription cannot be performed, thereby inhibiting transcription. Taking the fatty acid operon as an example, when we add arabinose, arabinose binds to the repressor protein on the arabinose operon and causes it to fall off. RNA polymerase is slid through the arabinose operon and slid to the downstream fatty acid operon to be repressed by a repressor protein on the fatty acid operon. At this time, we add fatty acids, and the fatty acids bind to the repressor protein, causing it to fall off. RNA polymerase continues to slide and slides over the fluorescent protein gene downstream of the operon to express the fluorescent protein.
Conversion：Introducing plasmid into competent cells
Set different inducer gradients

Observe the fluorescence concentration in the test tube every half hour

Detailed information about the experiment of "Preliminarily verify the function of the operon"can be found in Notebook-BUCT-China.

Results:

Fatty acid ec operon:Fluorescent protein expression of No. 1 tube and No. 2 tube, no fluorescence of No. 3 tube and No. 4 tube.
Glyoxylic acid operon:No. 1 tube and No. 4 tube have no fluorescence, and No. 2 tube and No. 3 tube have fluorescent expression
Fatty acid bh operon:No fluorescent expression was observed in No. 1 tube, No. 2 tube, No. 3 tube, and No. 4 tube.

More information about the Results can be found in Results--BUCT-China .

Quantitatively verify the function of the operon

Principle : The experimental principle is basically the same as the qualitative experiment.
The difference is that we can establish the mathematical model by measuring the fluorescence value of the culture under the same conditions of the OD value of each culture.Fluorescence value is proportional to the relationship between time.
Experimental protocol: For the fatty acid operon, we designed four control groups to measure the fluorescence value. The first group only added arabinose; the second group added different concentrations of fatty acid after adding arabinose for one hour; The group only added gradient fatty acids; the fourth group was a blank control group.
Stage results: The data on the right is the fluorescence value measured for the first three hours after eight hours of bacterial culture. From this set of data, it can be seen that the fluorescence value of the second group is higher than that of the first group and the blank control group, indicating that the fatty acid operon plays a role.
Follow-up experiment: We want to knock out the Fade gene in E. coli in subsequent experiments to eliminate the effect of this gene on the detection of fluorescence. This experiment is in progress.

Principle : The experimental principle is basically the same as the qualitative experiment.
The difference is that we can establish the mathematical model by measuring the fluorescence value of the culture under the same conditions of the OD value of each culture.Fluorescence value is proportional to the relationship between time.
Experimental protocol: For the glyoxylic acid operon, we designed four sets of control experiments to measure the fluorescence value, the first group only added arabinose; the second group added glyoxylic acid after one hour of arabinose reaction; the third group B Aldehydic acid; the fourth group was a blank control group.
Stage results: The data on the right is the fluorescence value measured for the first three hours after eight hours of bacterial culture. From this set of data, it can be seen that the fluorescence value of the second group is higher than that of the first group and the blank control group, indicating that the glyoxylic acid operon plays a role.

Citation

[1]Linda J. Wallace,Gary Wilcox. Regulation of the L-arabinose operon in strains of Escherichia coli containing ColE1-ara hybrid plasmids[J]. MGG Molecular & General Genetics,1979,173(3).