Parts test results

1. ara+RBS+BTE+Term

Since we need to produce free fatty acids by BTE in a controlled way, we have to determine the expression level of BTE on the plasmid we transferred into the E.coli and the most appropriate concentration of L-Arabinose to induce the BTE gene.

Expression of them was confirmed by quantitative PCR (qPCR) analysis of transformed BL21 E.coli collected from the Luria-Bertani broth. After 1:100 inoculation for 2.5 hour, the plates with E.coli had been induced stayed in the 37℃ incubator overnight. 5 and 10 mmol/L are the concentration of L-Arabinose we used to induce the ara operon, while the control group was the plate with no L-Arabinose.

Figure 1. RT-qPCR showed that plasmid successfully express BTE in E.coli.

We are delighted to notice that the BTE gene has a highly-expressed level.We found that the more L-Arabinose we give ,the higher expression of BTE E.coli have. We speculate that as the concentration of L-Arabinose increases, the BTE gene expresses higher. The result meets our expectation. We believe that the plasmid we transformed into the E.coli functions indeed. We chose 5 and 10 mmol/L as the appropriate concentration after many experiments, and finally we found that the range between 5 and 10 mmol/L is the most suitable situation to induce the plasmid.

In the mixture, we obtained 5 kinds of fatty acid, namely fatty acid of 10C chain length, 12C chain length, 14C chain length, 16C chain length and 18C chain length. Among them, fatty acid of 12C chain length accounts for the greatest amount of 74.2% and fatty acid of 10C chain length is the least amount of 1%. Proportion of fatty acid of 14C, 16C and 18C is 12.9%, 8% and 3.9% respectively.

2. lac+RBS+AtFatA+Term

Expression of them was confirmed by quantitative PCR (qPCR) analysis of transformed BL21 E.coli collected from the M9 Medium Broth. After 1:50 inoculation for 4 hour, the plates with E.coli stayed in the 37℃ incubator overnight. 8 and 16 g/L are the concentration of L-Arabinose we used to induce the lac operon, while we didn’t induce it in the control group.

Figure 2. RT-qPCR showed that plasmid successfully express AtfatA in E.coli.

We are delighted to notice that the BTE gene has a highly-expressed level. We found that the more lactose we give ,the higher expression of AtFatA E.coli have. We speculate that as the concentration of lactose increases, the AtfatA gene expresses higher.

The result makes us believe that the plasmid we transformed into the E.coli functions indeed. We chose 8 and 16 g/L as the appropriate concentration after many experiments, and finally we found that the range between 8 and 16 g/L is the most suitable situation to induce the plasmid. The higher or lower concentration will influence the expression of gene either.

This diagram is the gas chromatogram of the fatty acid produced by BL21 wild-type.

Total concentration of fatty acid is 0.141mg/L

This diagram is the gas chromatogram of the fatty acid produced by overexpressing AtFatA gene.It can be easily demonstrated that every kinds of fatty acid is greatly produced and among them fatty acid of 16C chain length is the most.

Total concentration of fatty acid is 20.3mg/L

In the mixture, we obtained 6 kinds of fatty acid, namely fatty acid of 14C chain length, 15C chain length with double-band, 16C chain length, 17C chain length with double-band, 18C chain length and 18C chain length with double-band. Among them, fatty acid of 16C chain length accounts for the greatest amount of 45.07% and fatty acid of 18C chain length is the least amount of 4.04%. Proportion of fatty acid of 14C, 15C with double-band, 17C with double-band and 18C with double-band is 5.61%, 7.96%, 16.21% and 21.11% respectively.

3. FabAB

This diagram is the gas chromatogram of the fatty acid produced by wild type BL21 and we can found that there are a few kinds of fatty acids in the whole product.

In the mixture, we mainly obtained 5 kinds of fatty acid, namely fatty acid of 14C chain length, 15C chain length, 16C chain length with double-band, 18C chain length and 18C chain length with double-band. Proportion of fatty acid of 14C chain length, 15C chain length, 16C chain length with double-band, 18C chain length and 18C chain length with double-band is 7.03%, 48.02%, 20.80%, 11.77% and 12.38% respectively.

After we gained the exhilarating result, we use the fermentor to culture the e.coli. We believe that lab environment’s is different with the fermentor’s one. And the fermentor’s result speaks louder than the lab’s one! As good as anticipated, the long chain fatty acid expressed in a high level. We did it!