Difference between revisions of "Part:BBa K2062006:Design"

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2062006 short</partinfo>
 
<partinfo>BBa_K2062006 short</partinfo>
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<html>
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<hr>
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<figure>
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  <img src="https://static.igem.org/mediawiki/2016/a/a1/Rhamnolipid-pathway.jpg"
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      alt="Rhamnolipid Pathway" width="500">
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  <figcaption>Figure 1: Metabolic pathway of <em>Pseudomonas
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  aeruginosa</em>.</figcaption>
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</figure>
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<p>
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  Rhamnolipids are naturally synthesized by the skin bacteria
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  <em>Pseudomonas aeruginosa</em> using the metabolic pathway
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  illustrated in Figure 1.
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</p>
 +
  <h2>Transformation of <em>P. putida</em> KT2440</h2>
 +
<p>
 +
  In order to avoid the virulence factors of <em>Pseudomonas
 +
  aeruginosa</em>, bacterial strains with similar or shared
 +
  metabolic pathways to the one above were chosen as potential
 +
  candidates.  The final candidates were <em>Pseudomonas putida</em>
 +
  and <em>Staphylococcus epidermidis</em>. Although <em>S. epidermidis</em>
 +
  doesn't share the same exact pathway as <em>P. aeruginosa</em>, it is
 +
  a naturally-occurring skin microbiome and only need two
 +
  additional enzymes, RhlA and RhlB, to produce
 +
  mono-rhamnolipids.  rhlA and rhlB genes necessary for
 +
  mono-rhamnolipid synthesis were extracted from the
 +
  <em>P. aeruginosa P14</em> bacterial strain to be placed into the
 +
  modified plasmid pNJ3.1 for transformation into the desired
 +
  bacterial strains (Figure 2).  The plasmid pC194 and a
 +
  shuttle vector strain, <em>S. aureus RN4220</em>, were used for <em>S. epidermidis</em>
 +
  transformations with the same basic design (Figure 2).
 +
</p>
 +
<p>
 +
  The plasmid pNJ3.1 has a promoter library that includes
 +
  hundreds of constitutive promoters with the length of 180
 +
  base pairs taken from various microbiome.  It is located at
 +
  the upstream of the gene that codes for super-folded GFP,
 +
  and the expression level of each constitutive promoter is
 +
  quantified with the intensity of fluorescence excited at 480
 +
  nm and emitted at 511 nm.
 +
</p>
  
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<partinfo>BBa_K2062006 SequenceAndFeatures</partinfo>
 
  
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<figure>
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  <img src="https://static.igem.org/mediawiki/parts/d/db/Circuit_rhlC.png"
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      alt="Circuit" width="500">
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  <figcaption>Figure 2: Circuit Design</figcaption>
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</figure>
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</hr>
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</html>
  
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===Source===
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<partinfo>BBa_K2062006 SequenceAndFeatures</partinfo>
−
 
+
−
Pseudomonas Aeruginosa P14
+

Latest revision as of 06:03, 24 October 2016


rhamnosyltransferase 2 [Pseudomonas aeruginosa]

Rhamnolipid Pathway
Figure 1: Metabolic pathway of Pseudomonas aeruginosa.

Rhamnolipids are naturally synthesized by the skin bacteria Pseudomonas aeruginosa using the metabolic pathway illustrated in Figure 1.

Transformation of P. putida KT2440

In order to avoid the virulence factors of Pseudomonas aeruginosa, bacterial strains with similar or shared metabolic pathways to the one above were chosen as potential candidates. The final candidates were Pseudomonas putida and Staphylococcus epidermidis. Although S. epidermidis doesn't share the same exact pathway as P. aeruginosa, it is a naturally-occurring skin microbiome and only need two additional enzymes, RhlA and RhlB, to produce mono-rhamnolipids. rhlA and rhlB genes necessary for mono-rhamnolipid synthesis were extracted from the P. aeruginosa P14 bacterial strain to be placed into the modified plasmid pNJ3.1 for transformation into the desired bacterial strains (Figure 2). The plasmid pC194 and a shuttle vector strain, S. aureus RN4220, were used for S. epidermidis transformations with the same basic design (Figure 2).

The plasmid pNJ3.1 has a promoter library that includes hundreds of constitutive promoters with the length of 180 base pairs taken from various microbiome. It is located at the upstream of the gene that codes for super-folded GFP, and the expression level of each constitutive promoter is quantified with the intensity of fluorescence excited at 480 nm and emitted at 511 nm.

Circuit
Figure 2: Circuit Design


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 622
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 205
    Illegal NgoMIV site found at 393
    Illegal NgoMIV site found at 931
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 664
    Illegal BsaI.rc site found at 898