Team:ETH Zurich/PABA/Results

From 2012.igem.org

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Figure2: HPLC output (290nm) Left: Standard disolved in acetonitril; Right: Construct in acetonitril.  
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Figure2: HPLC output (290nm) Left: Standard dissolved in acetonitril; Right: Construct in acetonitril.  
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As a control we used the wiltype Top10. Due to the fact that all bacteria produce PABA as an intermediate of their general biosynthesis, we also expect a signal in this sample. In Figure 1 we see a peak with a retention time of approximately 2 minutes. To be sure that this is the peak of PABA we also used pure PABA dissolved in acetonitril (Figure2), which also shows a retention time of 2 minutes. This graph also shows a second peak, which we could explain with the dissolvent. Dissolved in water we get a a single peak with a retention time of approximately 4 minutes.  
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As a control we used the wildtype Top10. Due to the fact that all bacteria produce PABA as an intermediate of their general biosynthesis, we also expect a signal in this sample. In Figure 1 we see a peak with a retention time of approximately 2 minutes. To be sure that this is the peak of PABA we also used pure PABA dissolved in acetonitril (Figure2), which also shows a retention time of 2 minutes. This graph also shows a second peak, which we could explain with the dissolvent. Dissolved in water we get a a single peak with a retention time of approximately 4 minutes.  
To rule out the possibility that this peak comes from the dissolvent itself, we also run acteonitril, which we used to lyse our cells. This control showed no peak.
To rule out the possibility that this peak comes from the dissolvent itself, we also run acteonitril, which we used to lyse our cells. This control showed no peak.
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The amount of PABA matches the same concentration as the wildtype. This suggests that our construct could not accumulate PABA in the cell.   
The amount of PABA matches the same concentration as the wildtype. This suggests that our construct could not accumulate PABA in the cell.   
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PABA is not accumulated in the cell, maybe due to the fact that not enough chorismate is in the cell. Therefore we are currently trying to work with a E.coli strain called K12 <span class="eth_reference">[Kast1996]</span>, which accumulates Chorismate in the cell due to a deficiency in the two Chorismate mutases. Since chorismate is a precursor molecule we hope to increase the PABA output.  
+
PABA is not accumulated in the cell, maybe due to the fact that not enough chorismate is in the cell. Therefore we are currently trying to work with an ''E.coli'' K12 chorismate overproducer strain <span class="eth_reference">[Kast1996]</span>, which accumulates chorismate in the cell due to a deficiency in two chorismate mutases. Since chorismate is a precursor molecule we hope to increase PABA output.  
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Revision as of 02:21, 27 October 2012

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Navigate through Overview, Design and Results

Contents

PABA generator

Cloning

Fig.1: Plasmid map of <partinfo>BBa_K909014</partinfo> in pSB1C3.

We were succesful in constructing both parts <partinfo>BBa_K909014</partinfo> (2699bp) and <partinfo>BBa_K909015</partinfo> (2656bp) based on the already existing parts <partinfo>BBa_K137055</partinfo> and <partinfo>BBa_S04039</partinfo>. The vector backbone is <partinfo>pSB1C3</partinfo> (2079bp). They differ from each other due to the fact that <partinfo>BBa_K909014</partinfo> contains the constitutive promoter <partinfo>BBa_J23100</partinfo> with two NheI restriction sites.

To verify the sizes of the constructs, the plasmids containing <partinfo>BBa_K909014</partinfo> and <partinfo>BBa_K909015</partinfo> were digested with

1. NheI & PstI

  • Expected bands of <partinfo>BBa_K909014</partinfo>: 2685 bp & 2061 bp
  • Expected bands of <partinfo>BBa_K909015</partinfo>: 4724 bp

2. XbaI & PstI

  • Expected bands of <partinfo>BBa_K909014</partinfo>: 2721 bp & 2048 bp
  • Expected bands of <partinfo>BBa_K909015</partinfo>: 2678 bp & 2048 bp
Fig.2:Plasmid map of <partinfo>BBa_K909015</partinfo> in pSB1C3.


Fig.3.: Digests left to right: Ladder, <partinfo>BBa_K909014</partinfo> (NheI/PstI), <partinfo>BBa_K909014</partinfo> (XbaI,PstI),<partinfo>BBa_K909015</partinfo> (NheI/PstI), <partinfo>BBa_K909015</partinfo> (XbaI,PstI).


As it is visible in Fig.3. the band sizes of the <partinfo>BBa_K909014</partinfo> & <partinfo>BBa_K909015</partinfo> digestion match the expectation. In a further step <partinfo>BBa_K909015</partinfo> is joined to the PL hybrid promoter <partinfo>BBa_K909011</partinfo> for implementation.


Enzyme overexpression

We were able to overexpress the enzymes pabA, pabB and pabC with our construct. We showed this with an SDS-gel compared to a WT sample. We clearly see a band of the size 22 kDa (pabA) and 71 kDa (pabB/C) which is bigger compared to the WT.


PABA overproduction

We used high-performance liquid chromatography to determine the amount of PABA in our cells. We performed a standard curve with pure PABA. PABA was dissolved as well as the samples in acteonitril and applied to the HPLC.

Wt.png
Acetonitril.png

Figure1: HPLC output (290nm) Left: wildtype in acetonitril; Right: Acetonitril without a sample.

Sta.png
Ab.png

Figure2: HPLC output (290nm) Left: Standard dissolved in acetonitril; Right: Construct in acetonitril.

As a control we used the wildtype Top10. Due to the fact that all bacteria produce PABA as an intermediate of their general biosynthesis, we also expect a signal in this sample. In Figure 1 we see a peak with a retention time of approximately 2 minutes. To be sure that this is the peak of PABA we also used pure PABA dissolved in acetonitril (Figure2), which also shows a retention time of 2 minutes. This graph also shows a second peak, which we could explain with the dissolvent. Dissolved in water we get a a single peak with a retention time of approximately 4 minutes.

To rule out the possibility that this peak comes from the dissolvent itself, we also run acteonitril, which we used to lyse our cells. This control showed no peak.

Figure 2 on the right shows our construct, overexpressing pabA, pabB and pabC. We expect a higher amount of PABA in this sample, an accumulation of PABA. The amount of PABA matches the same concentration as the wildtype. This suggests that our construct could not accumulate PABA in the cell.

PABA is not accumulated in the cell, maybe due to the fact that not enough chorismate is in the cell. Therefore we are currently trying to work with an E.coli K12 chorismate overproducer strain [Kast1996], which accumulates chorismate in the cell due to a deficiency in two chorismate mutases. Since chorismate is a precursor molecule we hope to increase PABA output.


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