We designed 4 sets of plasmids and selected [pBbE8a-RFP] plasmid as the master plate for transformation. The No. 1, No. 3, and No. 4 plasmids were loaded with fatty acid operons, glyoxylate operons and another fatty acid operons, respectively. The No. 2 plasmid was used as a vector plasmid to replace the white segment with the arabinose operon, which later reached the role of the vector plasmid. After that, we sent the designed four sets of plasmid information to addgen for plasmid synthesis.

2018.1

We transformed the plasmid into competent cells, cultured and proliferated, and preserved. The plasmid was extracted and subjected to DNA electrophoresis verification, that is, qualitative verification was performed.

Next, the plasmid was digested. At the start and end of our operon, there were Bgl Ⅱ and Xho Ⅰ cleavage sites, respectively. Therefore, the corresponding restriction enzyme was used for digestion. After that, the enzyme-cut products were subjected to DNA electrophoresis, and each set of plasmids was cut into two sections, which corresponded to two strips on the electrophoresis gel on the electrophoresis gel. Thereafter, a gel recovery operation was carried out, and for the first, third, and fourth groups of plasmids, a band having a molecular weight of about 1,700 was recovered, which was the intended operon.

For the second group, a band having a molecular weight of about 4,000 was recovered to serve as a carrier. The recovered plasmid fragments are then ligated by ligating the target operons obtained in groups 1, 3, and 4 with the vector plasmid fragments of the two groups to form a new plasmid. The next step is to transfer the target plasmid to competent bacteria for a new round of plasmid amplification.

We extracted the plasmids in bacteria No. 1, 3 and 4, and cut the plasmid with the enzymes Bgl II and Xho I, and then verified by electrophoresis.It was found that all the molecular weights in 1, 3 and 4 are divided into two strips ,the molecular weight of a strip is 1700 and another one is 4000. It can be verified that our previous digestion and plasmid ligation operations are correct.

2018.2

We verified the function of the operon, and set up 4 sets of control experiments for each plasmid. The specific experimental conditions are as follows:

Arabinose: Add 20% arabinose solution
Fatty acid: Add 100mmol/L fatty acid solution
Glyoxylic acid: Add 2mol/L glyoxylic acid solution

Detailed information about the January and February experiment can be found in the documentation: BUCT--China--Notebook.

2018.7

In July, We performed a fluorescence test on the plasmid and completed the Interlab experiment.

Fluorescence detection of fatty acid operons, we want to use the E. coli knockout of the fade gene to accurately detect the fluorescence value, but unfortunately, our knockout and reconstruction work has not been completed, so our original bacteria measured fluorescence value For details, please refer to:BUCT-China/Experiments .

For the fluorescence detection of the glyoxylic acid operon, we used the plasmid that has been successfully constructed and introduced into E. coli for quantitative determination. The data showed that the glyoxylic acid operon played a role. For details, please see:BUCT-China/Experiments .

More information about Interlab can be found in Interlab--BUCT-China .

Team:BUCT-China/Notebook

Notebook

Detailed information about the January and February experiment can be found in the documentation: BUCT--China--Notebook.