Team:BIOTEC Dresden/ACP

From 2010.igem.org

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     <p>  If we substitute in the reaction above CoA with derivatives containing the acyl group already attached to the phosphopantetheine moiety, then we would be able to produce hexanoyl-ACP (figure on the right) ready to be used directly for the <i> in vitro </i> the reaction with LuxI. </p>
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     <p>  If we substitute in the reaction above CoA with derivatives containing the acyl group already attached to the phosphopantetheine moiety, then we would be able to produce hexanoyl-ACP (figure on the right) ready to be used directly for the <i> in vitro </i> reaction with LuxI. </p>
<a href="http://2010.igem.org/wiki/images/6/66/ACP2.jpg"  rel="lightbox"><img src="http://2010.igem.org/wiki/images/6/66/ACP2.jpg" class="border thumb right"></a></p>
<a href="http://2010.igem.org/wiki/images/6/66/ACP2.jpg"  rel="lightbox"><img src="http://2010.igem.org/wiki/images/6/66/ACP2.jpg" class="border thumb right"></a></p>
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<a href="http://2010.igem.org/wiki/images/7/72/Butyryl-CoA.jpg"  rel="lightbox"><img src="http://2010.igem.org/wiki/images/7/72/Butyryl-CoA.jpg" class="border thumb right"></a></p>
  <p>  There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </p>
  <p>  There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </p>
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  </p>  The behavior of the enzyme depending on the substrate is illustrated in figure 3. </p>
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  </p>  The behavior of the ACP synthase depending on the CoA and apo-ACP substrates is illustrated in the figure below. </p>
<a href="http://2010.igem.org/wiki/images/9/9f/CoA%2BApo.jpg"  rel="lightbox"><img src="http://2010.igem.org/wiki/images/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p>
<a href="http://2010.igem.org/wiki/images/9/9f/CoA%2BApo.jpg"  rel="lightbox"><img src="http://2010.igem.org/wiki/images/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p>
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<a href="http://2010.igem.org/wiki/images/7/72/Butyryl-CoA.jpg"  rel="lightbox"><img src="http://2010.igem.org/wiki/images/7/72/Butyryl-CoA.jpg" class="border thumb right"></a></p>
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</p>    As a successful example from literature to support our experimental design, the Km value for butyryl production (fig. 4) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </p>
+
</p>    As a successful example from literature to support our experimental design, the Km value for butyryl production (figure on the right) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </p>
<h2>  Detection </h2>
<h2>  Detection </h2>
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<a href="http://2010.igem.org/wiki/images/2/2a/Gel222222.jpg"  rel="lightbox"><img src="http://2010.igem.org/wiki/images/2/2a/Gel222222.jpg" class="border thumb right"></a></p>
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   <p>  A first simple step to judge if the reaction is successful would be to run the reaction mixture on a gel. There is evidence that, in spite of a bigger mass, acylated ACP can travel faster than apo or holoACP in an SDS-PAGE, due to active binding of the SDS detergent molecules to the hydrophobic acyl chain which increases the charge to mass ratio of the entire molecule, thus, facilitating faster migration (fig. 5) (2,7). On the other hand it is possible to run the reaction mixture also on a native gel and compare the migration pattern of the proteins. The potein that would migrate faster than apoACP in SDS PAGE but slower than it in a native page (separation by mass) would be the acylated ACP (fig. 6). Supposing that just a fraction of the ACP interacts to produce the acylated derivative, bands for both hexanoyl-ACP and apo/ holoACP were also expected. Dimerization of holo-ACP would also generate and additional band) </p>   
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   <p>  A first simple step to judge if the reaction is successful would be to run the reaction mixture on a gel. There is evidence that, in spite of a bigger mass, acylated ACP can travel faster than apo or holo ACP in an SDS-PAGE, due to active binding of the SDS detergent molecules to the hydrophobic acyl chain which increases the charge to mass ratio of the entire molecule, thus, facilitating faster migration (figure on the right, length of acyl chains is indicated below, "-" stands for holo ACP, 6:0 - for hexanoyl-ACP) (2,7). On the other hand it is possible to run the reaction mixture also on a native gel and compare the migration pattern of the proteins. The potein that would migrate faster than apoACP in SDS PAGE but slower than it in a native page (separation by mass) would be the acylated ACP. Supposing that just a fraction of the ACP interacts to produce the acylated derivative, bands for both hexanoyl-ACP and apo/ holoACP were also expected. Dimerization of holo-ACP would also generate a band) </p>   
<p>      Although this approach isn’t quantitative, it would give a hint about the efficiency of the reaction. </p>   
<p>      Although this approach isn’t quantitative, it would give a hint about the efficiency of the reaction. </p>   
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<p>      An intermediary step for the detection of the acyl-ACP synthesis IS the incubation of the reaction mixture with cell extracts of strains constitutively expressing LuxI. Using as a control LuxI extract incubated with a receiver strain and no reaction mixture, normalization is possible and the relation of fluorescence to the amount of reaction mix provided can be tracked.  </p>  
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<p>      An intermediary step for the detection of the acyl-ACP synthesis is the incubation of the reaction mixture with cell extracts of strains constitutively expressing LuxI. Using as a control LuxI extract incubated with SAM, a receiver strain and no reaction mixture, normalization is possible and the relation of fluorescence to varying amounts of the provided reaction mix containing hexanoyl-ACP  can be tracked. Quantification can be done to assess the maximally efficient conditions for acyl-ACP synthesis. </p> </p>  
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<p>    A next integrating approach is to test our system with that of the “Fusion protein” (yellow team). If we test our AHL-sensitive reporter cells with SAM, hexanoyl-ACP containing reaction mixture and the recombinant LuxI, then the activation of the reporter cells by this reaction mixture proves the fact that we have produced both functional acyl-ACP and preserved the AHL producing function of the fusion protein. More than this, quantification can be done to assess the maximally efficient conditions for acyl-ACP synthesis </p>
+
<p>    A next integrating approach is to test our system with that of the “Fusion protein”. If we test our AHL-sensitive reporter cells again with SAM, hexanoyl-ACP containing reaction mixture and the recombinant LuxI, then the activation of the reporter cells by this reaction mixture proves the fact that we have produced both functional acyl-ACP and preserved the AHL producing function of the fusion protein (<i>in-vitro</i> activity proved).
<h2> Methods </h2>
<h2> Methods </h2>
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  <p>    The reaction components and their molar ratios were adjusted based on previous studies (4,5) and are as follows: 50 mM Tris-HCl, ph 8.0 buffer, 10 mM MgCl2 (enzyme cofactor), 1 mM DTT, 300 µM CoA, 50 µM of apo-ACP and 5 µM of ACP synthase in a total of 100µl reaction volume. The mix is incubated at 37°C for 30 minutes. For terminating the reaction, 50 mM EDTA or 10% trichloroacetic acid is added. </p>
  <p>    The reaction components and their molar ratios were adjusted based on previous studies (4,5) and are as follows: 50 mM Tris-HCl, ph 8.0 buffer, 10 mM MgCl2 (enzyme cofactor), 1 mM DTT, 300 µM CoA, 50 µM of apo-ACP and 5 µM of ACP synthase in a total of 100µl reaction volume. The mix is incubated at 37°C for 30 minutes. For terminating the reaction, 50 mM EDTA or 10% trichloroacetic acid is added. </p>
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<h2> References </h2>
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References are listed in the Resource/ Literature section
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Latest revision as of 03:00, 28 October 2010

ACP

ABREVIATIONS: ACP – acyl carrier protein, SAM – S-adenosyl-methionine, CoA – coenzyme A

Aim

For enabling the outside-of-cell activity of the recombinant LuxI enzyme which will serve as the signal transducing element in our system we needed to provide the substrates for it: S-adenosyl-methionine (SAM); which also serves as a universal donor in methylation reactions and is easy to get from the store and acylated-acyl carrier protein (in our case hexanoyl-ACP); which is not readily available commercially but is possible to synthesize by more available strategies. The synthesis and detection of this compound is discussed further.

Synthesis

There are two major quorum signaling molecules produced by the V. fischeri LuxI enzyme in almost equimolar concentrations: N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone (1). The two compounds differ only by a keto group at the third carbon of the acyl chain, but hexanoyl homoserine lactone was used since it was advantageous to try synthesizing it using commercially available products.

Normally the in vivo synthesis of the acyl-ACP substrate for the production of signaling molecules in bacterial quorum sensing is carried out by enzymes like acyl-ACP synthetases which can transfer fatty acid groups onto the functional ACPs (already containing the phosphopantetheine prosthetic group). The two-step purification of the V. fischeri LuxI enzyme and in vitro ACP acylation reactions have been achieved before (2).

We, however, decided to try an entirely different synthesis approach by using a different enzyme, namely ACP synthase (4-phosphopantetheinyl-transferase) which is involved in a more general reaction that spans all the domains of life and represents the addition of a prosthetic group to an ACP protein either from the fatty acid or polyketide synthesis pathways in order to make it functional. The prosthetic group in this case is a phosphopantetheine which is taken directly from CoA and added to the apo-protein variant of ACP generating adenosine 3',5'-bisphosphate and holo-ACP (reaction depicted on the right side).

If we substitute in the reaction above CoA with derivatives containing the acyl group already attached to the phosphopantetheine moiety, then we would be able to produce hexanoyl-ACP (figure on the right) ready to be used directly for the in vitro reaction with LuxI.

In fact, the described reaction is a well-established commercial technique used for labeling cell surface proteins fused to ACP with acyl groups of various lengths. (3)

We tried to produce the reaction using 3 main players:

  1. Hexanoyl CoA trilithium salt hydrate
  2. Acyl carrier protein HPLC from E.coli – as an alternative for this compound, purified ACP from an E.coli ACPS mutant strain (overexpressing apo-ACP) can be used (8)
  3. ACP synthase from E.coli

There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6)

The behavior of the ACP synthase depending on the CoA and apo-ACP substrates is illustrated in the figure below.

As a successful example from literature to support our experimental design, the Km value for butyryl production (figure on the right) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times.

Detection

A first simple step to judge if the reaction is successful would be to run the reaction mixture on a gel. There is evidence that, in spite of a bigger mass, acylated ACP can travel faster than apo or holo ACP in an SDS-PAGE, due to active binding of the SDS detergent molecules to the hydrophobic acyl chain which increases the charge to mass ratio of the entire molecule, thus, facilitating faster migration (figure on the right, length of acyl chains is indicated below, "-" stands for holo ACP, 6:0 - for hexanoyl-ACP) (2,7). On the other hand it is possible to run the reaction mixture also on a native gel and compare the migration pattern of the proteins. The potein that would migrate faster than apoACP in SDS PAGE but slower than it in a native page (separation by mass) would be the acylated ACP. Supposing that just a fraction of the ACP interacts to produce the acylated derivative, bands for both hexanoyl-ACP and apo/ holoACP were also expected. Dimerization of holo-ACP would also generate a band)

Although this approach isn’t quantitative, it would give a hint about the efficiency of the reaction.

An intermediary step for the detection of the acyl-ACP synthesis is the incubation of the reaction mixture with cell extracts of strains constitutively expressing LuxI. Using as a control LuxI extract incubated with SAM, a receiver strain and no reaction mixture, normalization is possible and the relation of fluorescence to varying amounts of the provided reaction mix containing hexanoyl-ACP can be tracked. Quantification can be done to assess the maximally efficient conditions for acyl-ACP synthesis.

A next integrating approach is to test our system with that of the “Fusion protein”. If we test our AHL-sensitive reporter cells again with SAM, hexanoyl-ACP containing reaction mixture and the recombinant LuxI, then the activation of the reporter cells by this reaction mixture proves the fact that we have produced both functional acyl-ACP and preserved the AHL producing function of the fusion protein (in-vitro activity proved).

Methods

The reaction components and their molar ratios were adjusted based on previous studies (4,5) and are as follows: 50 mM Tris-HCl, ph 8.0 buffer, 10 mM MgCl2 (enzyme cofactor), 1 mM DTT, 300 µM CoA, 50 µM of apo-ACP and 5 µM of ACP synthase in a total of 100µl reaction volume. The mix is incubated at 37°C for 30 minutes. For terminating the reaction, 50 mM EDTA or 10% trichloroacetic acid is added.

References

References are listed in the Resource/ Literature section

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