Team:NJU-QIBEBT/ACHIEVEMENT/Parts test results

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<p>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.
<p>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.
</p>
</p>
-
<p>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. 5,10 and 20 mmol/L are the concentration of L-Arabinose we used to induce the ara operon, while 0 means it was the control group.
+
<p>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.
</p>
</p>
-
<p>Figure 1. RT-qPCR showed that plasmid successfully express BTE in E.coli. The more L-Arabinose we give ,the higher expression of BTE E.coli have. To our surprise, when we used 20mmol/L L-Arabinose to induce the E.coli, the expression of BTE gene decreased a lot. We speculate that it is because the concentration of L-Arabinose is too high for the ara operon.
+
<img src="https://static.igem.org/mediawiki/2014/f/fb/Result2-qz_%281%29.png" >
 +
<p>Figure 1. RT-qPCR showed that plasmid successfully express BTE in E.coli.</p>
 +
<p>
 +
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.
</p>
</p>
-
 
+
<img src="https://static.igem.org/mediawiki/2014/3/36/Sdfgrdg.png" >
-
<h2>2. lac+FabA&B
+
<p>
 +
<br>
 +
The proportion of 12C/14C fatty acid produced by BTE and Wild Type
 +
<p>
 +
</p>
 +
<br>
 +
<p>
 +
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.
 +
</p>
 +
<img src="https://static.igem.org/mediawiki/2014/4/45/BTE.png">
 +
<br>
 +
The proportion of BTE products
 +
<p>
 +
</p>
 +
<br>
 +
<p>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.
 +
</p>
 +
<br><br/>
 +
<br><br/>
 +
<div class="hr_pro">
 +
<hr style="" width="100%" >
 +
<hr size="10px" noshade=true />
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</div>
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<h2>2. lac+RBS+AtFatA+Term
</h2>
</h2>
-
<p>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.
+
<p>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.
</p>
</p>
-
<p>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.
+
<img src="https://static.igem.org/mediawiki/2014/d/da/Result1-qz.png" >
 +
<p>Figure 2. RT-qPCR showed that plasmid successfully express AtfatA in E.coli.
</p>
</p>
 +
<p>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.
 +
</p>
 +
<p>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.
 +
</p>
 +
<img src="https://static.igem.org/mediawiki/2014/1/16/%E9%87%8E%E7%94%9F%E5%9E%8Bgrtyr.png" width="900px" >
 +
<p>
 +
This diagram is the gas chromatogram of the fatty acid produced by BL21 wild-type.
 +
</p>
 +
<p>
 +
Total concentration of fatty acid is  0.141mg/L
 +
</p>
 +
<img src="https://static.igem.org/mediawiki/2014/8/8d/AtFatAsdfgbsdgsdg.png" width="900px" >
-
<h2>3. lac+RBS+AtFatA+Term
+
<p>
 +
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.
 +
</p>
 +
 
 +
<p>
 +
Total concentration of fatty acid is 20.3mg/L
 +
</p>
 +
<p>
 +
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.
 +
</p>
 +
<img src="https://static.igem.org/mediawiki/2014/d/d9/AtFatAtekrjgisdug.png" >
 +
<p>
 +
The proportion of AtFatA products
 +
</p>
 +
<img src="https://static.igem.org/mediawiki/2014/4/4a/%E6%AF%94%E8%BE%83.png">
 +
<p>The product of AtFatA compared with Wild Type
 +
 
 +
</p>
 +
<p>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.
 +
</p>
 +
<br><br/>
 +
<br><br/>
 +
<div class="hr_pro">
 +
<hr style="" width="100%" >
 +
<hr size="10px" noshade=true />
 +
</div>
 +
<h2>3. FabAB
</h2>
</h2>
-
<p>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.
+
 
 +
<img src="https://static.igem.org/mediawiki/2014/2/24/Sfjhdfyhjfg.png" width="960px" >
 +
<br>
 +
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.
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2014/0/02/Dfjhfdhdf.png" >
 +
<p>The proportion of FabAB products
</p>
</p>
-
<p>Figure 3. RT-qPCR showed that plasmid successfully express AtfatA in E.coli.
+
<p>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.
 +
<p>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.
</p>
</p>
-
<p>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.
+
<p>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.
 +
<p>
 +
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!
</p>
</p>
-
<p>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.
+
<br><br/>
 +
<br><br/>
 +
<div class="hr_pro">
 +
<hr style="" width="100%" >
 +
<hr size="10px" noshade=true />
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</div>
 +
<h2>4.Fluorescence Report System</h2>
 +
<p>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.
</p>
</p>
-
<p>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!
+
<img src="https://static.igem.org/mediawiki/2014/1/19/System_design.png" >
 +
 
 +
<p></p>
 +
<img src="https://static.igem.org/mediawiki/2014/4/45/GFP_1.png" >
 +
<p></p>
 +
<img src="https://static.igem.org/mediawiki/2014/6/6b/RFP_1.png" >
 +
 
 +
 
 +
<p>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.
</p>
</p>
-
<h2>4.  T7+MsbA </h2>
+
<p>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.
-
<p>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.
+
</p>
</p>
-
<p>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.
+
 
 +
<p>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.
</p>
</p>
 +
<p>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.
 +
</p>
 +
 +
 +
<p>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.
 +
</p>
 +
<p></p>
<p></p>
</div>
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Latest revision as of 03:56, 18 October 2014

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.

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