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.

2. lac+FabA&B

Expression of FabA&B was confirmed by quantitative PCR (qPCR) analysis of transformed BL21 E.coli collected from the M9 Medium Broth. We inoculated the E.coli in the 1:50 proportion, and they stayed in the 37℃ incubator overnight. Then we used IPTG to induce it for 4 hours. 0.1,0.5 and 1 mmol/L are the concentration of IPTG we used to induce the lac operon, while 0 means it was the control group.

Figure 2. RT-qPCR showed that plasmid successfully express FabA&B in E.coli. The result indicated that after the IPTG induction, the expression of FabA&B decreased. We speculate that it is because IPTG has cytotoxicity and it can damage the E.coli in a certain degree. Still, we can prove that FabA&B gene in our E.coli had a much higher level of expression than the one in the wild type.

3. 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. 4,8 and 16 g/L are the concentration of L-Arabinose we used to induce the lac operon, while 0 means it was the control group.

Figure 3. RT-qPCR showed that plasmid successfully express AtfatA in E.coli.

Surprisingly, the plasmid expressed more AtFatA when there is no lactose and the least when the concentration is 8 g/L. At first, we speculated that the lac operon went wrong. Before we changed several lac operon, we chose M9 instead of Luria-Bertani broth because we could control the ingredients. Still, the result remained unchange.

We of not reconciled to still were holding tried state of mind in the arms to culture our E.coli in the fermentor. We were pleasantly surprised to find that the plasmid expressed the long chain fatty acid in a high level.

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!

4. T7+MsbA

Expression of MsbA 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 stayed in the 37℃ incubator overnight.

The result of q-PCR showed that the expression of MsbA increased about eight times. Due to the toxicity of T7 promoter to the E.coli, we cannot overexpress MsbA too much. We believe that the expression level of MsbA we got is completely enough to transport the free fatty acid.