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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.

The proportion of 12C/14C fatty acid produced by BTE and Wild Type

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.

The proportion of BTE products

Conclusion:We can infer from data that after induced by the L-Arabinose, the BTE gene achieve its function and the proportion of 12C/14C fatty acid rise significantly when compared with wild type. 12C fatty acid rise about 70% and 14C fatty acid rise about 10% which is almost twice proportion of the wild type.

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.

The proportion of AtFatA products

The product of AtFatA compared with Wild Type

Conclusion:These figures and diagrams show the fact that the proportion of fatty acid of 16C chain length and 18C chain length rise greatly after induced by lactose. Fatty acid of 16C chain length takes the most proportion of 45.07% in the whole project. Besides that, the production of fatty acid rise from 0.141mg/L to 20.3mg/L. It can indicate that the induce of lactose is successful and the function of AtFatA can be fulfilled.

3. FabAB

This diagram is the gas chromatogram of the fatty acid produced by overexpressing both FabA gene and FadB gene. We can found that there are a few kinds of fatty acids in the whole product.

The proportion of FabAB products

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.

Conclusion:We can find that the amount of fatty acid of 16C and 18C with double-band rose when compared with the wild type. The function of gene FabAB works.

Final conclusion: When compared with the control group which is the wild type BL21, we can find that the proportion of 16C/18C fatty acid rises because of the proportion of AtFatA, 12C/14C fatty acid rises due to the control of BTE and the proportion of unsaturated fatty acid rises because of the promotion of FabAB. Besides the proportion, the production of each kind of fatty acid also increase. These fact can indicate that the Gene AtFatA, BTE and FabAB really work and we successfully control the species of fatty acid by applying different signals as we expected.

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!

4.Fluorescence Report System

This system uses the most important transcriptional regulatory factor FadR in the synthesis and degradation process as the core and promoter FabB and FadD, named PFabB and PFadD hereinafter. The whole design is shown in the chart below: GFP is connected to the downstream of PFabB, which turns on the expression of GFP; RFP is connected to the downstream of PFadD, which turns on the expression of RFP.

When E.coli is inoculated on the fresh LB medium, the concentration of fatty acid is very low. E.coli is vigorously dividing which requires large amount of fatty acid as the source of membrane phospholipid. In this case, FadR binds to PFabB and PFadD to initiates the pathway of the synthesis of fatty acid and represses the degradation of it so that GFP is expressed while RFP is not.

As incubation time increases, the synthesis of fatty acid increases too. When E.coli develop into stable phase(about 6 hours), the bacteria stops or slows down to divide while the content of fatty acid gradually reaches saturation manifesting as the GFP expression peaks and starts to decrease. In another aspect, fatty acid turns into long chain acyl-CoA, which binds to FadR and fatty acid is from PFadD and PFabB, starting the expression of RFP.

As incubation time increases, the synthesis of fatty acid increases too. When E.coli develop into stable phase(about 6 hours), the bacteria stops or slows down to divide while the content of fatty acid gradually reaches saturation manifesting as the GFP expression peaks and starts to decrease. In another aspect, fatty acid turns into long chain acyl-CoA, which binds to FadR and fatty acid is from PFadD and PFabB, starting the expression of RFP.

In fact, the binding constant of FadR and acyl-CoA is variable which means that the expression of RFP and GFP isn’t absolute. Neither GFP nor RFP expresses itself alone. The chart below shows the expression level of GFP and RFP in the initial 24 hours.

As we can see, RFP increases along with the increase of GFP but GFP peaks ahead of RFP, which means fatty acid degrades as it increases. The degradation of fatty acid always falls behind its increase which is manifested in the chart as the lagging of RFP expression compared with GFP.In the meanwhile, the decrease of fatty acid maintains at a relatively low level, shown in the chart as the expression of RFP lower than GFP.