Difference between revisions of "Part:BBa K1655000"
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<partinfo>BBa_K1655000 short</partinfo>
 
<partinfo>BBa_K1655000 short</partinfo>
 
<html>
 
<html>
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<figure style="float:right;margin-left:3%;">
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  <img src="https://static.igem.org/mediawiki/2015/7/7c/Aalto-Helsinki_plasmid_propane1_for_white_bckgr.png" style="width:230px;"/>
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  <figcaption><b><center>Figure 1.</b> Propane 1</center></figcaption>
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</figure>
 
<p>Propane 1 includes 1)acyl-CoA thioester hydrolase (YciA) from Haemophilus influenzae, which catalyzes the reaction: acyl-CoA + H2O <-> CoA + a carboxylate, 2) Carboxylic acid reductase (CAR) from Mycobacterium marinum (strain ATCC BAA-535 / M), which catalyzes the reaction:  a carboxylate + reduced acceptor <-> an aldehyde + acceptor + H2O, and 3) maturation factor phosphopantetheinyl transferase (sfp) from Bacillus subtilis, which activates CAR by catalyzing the transfer of a  prosthetic phosphopanteine group to CAR.</p>
 
<p>Propane 1 includes 1)acyl-CoA thioester hydrolase (YciA) from Haemophilus influenzae, which catalyzes the reaction: acyl-CoA + H2O <-> CoA + a carboxylate, 2) Carboxylic acid reductase (CAR) from Mycobacterium marinum (strain ATCC BAA-535 / M), which catalyzes the reaction:  a carboxylate + reduced acceptor <-> an aldehyde + acceptor + H2O, and 3) maturation factor phosphopantetheinyl transferase (sfp) from Bacillus subtilis, which activates CAR by catalyzing the transfer of a  prosthetic phosphopanteine group to CAR.</p>
 +
 +
<p>Propane 1 can be used as a part to produce propane in <i>E. coli</i>. The propane production pathway is shown below, and the enzymes included in Propane 1 are highlighted in blue.</p>
 +
<figure style="float:left">
 +
  <img src="https://static.igem.org/mediawiki/2015/8/81/Aalto-Helsinki_propane1_enzymes_highlighted.png" style="width:900px;"/>
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</figure>
 +
  
 
<p>All coding regions are assembled as an operon after a T7 promoter and and RBS from common cloning vectors (such as pCDF-Duet1 by Novagen). Terminator sequence after the last coding sequence, CAR is also derived from common cloning vectors (e.g. pCDF-Duet1 by Novagen uses this terminator).</p>
 
<p>All coding regions are assembled as an operon after a T7 promoter and and RBS from common cloning vectors (such as pCDF-Duet1 by Novagen). Terminator sequence after the last coding sequence, CAR is also derived from common cloning vectors (e.g. pCDF-Duet1 by Novagen uses this terminator).</p>
  
 
<p>All coding sequences have been codon optimized twice for E. coli. First with Thermo Fisher's codon optimizer and then with IDT's codon optimization software.</p>
 
<p>All coding sequences have been codon optimized twice for E. coli. First with Thermo Fisher's codon optimizer and then with IDT's codon optimization software.</p>
 +
 +
<p>The plasmid has been assembled from IDT's gBlocks with NEBuilder assembly, similar to Gibson Assembly.</p>
 +
<p><b>Validation:</b> We restricted our Propane 1 and ran the insert on an agarose gel. From the picture we can tell that the insert's size is correct. The result can be seen in <a href="https://static.igem.org/mediawiki/2015/3/3a/Aalto-Helsinki_car_submittedparts.png">Figure 2.</a><br/>
 +
Additionally, we did a colony PCR with VR and our primer P001. The VR primer attaches to our plasmid's backbone while P001 anneals with the very beginning of our construct. With this colony PCR we were able to show that the insert is present and it is indeed in the pSB1C3 backbone. See <a href="https://static.igem.org/mediawiki/2015/d/d1/Aalto-Helsinki_submittedparts_colony_pcr_validation.png">figure 3</a> for results, where the product in wells 1, and 5-10 is of the right size.</p>
 +
<p>The colony which produced the product seen in figure 3, well 10 was sent to the registry.</p>
 +
<p>Click <a href="https://static.igem.org/mediawiki/2015/9/93/Aalto-Helsinki_car_sequence.gb">here</a> to download the full sequence of Propane 1 in pSB1C3 backbone.</p>
 +
 +
<p>Click the images to enlarge them.</p>
 +
<figure style="float:left; margin-right:2%;">
 +
  <a href="https://static.igem.org/mediawiki/2015/3/3a/Aalto-Helsinki_car_submittedparts.png"><img src="https://static.igem.org/mediawiki/2015/3/3a/Aalto-Helsinki_car_submittedparts.png" style="width:150px;"/></a>
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  <figcaption><b><center>Figure 2.</b> Restriction digestion</center></figcaption>
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</figure>
  
  
−
<p>Propane 1 can be used as a part to produce propane in <i>E. coli</i>. The propane production pathway is shown below, and the enzymes included in Propane 1 are highlighted in blue.</p>
 
 
<figure style="float:left">
 
<figure style="float:left">
−
   <img src="https://static.igem.org/mediawiki/2015/8/81/Aalto-Helsinki_propane1_enzymes_highlighted.png" style="width:900px;"/>
+
   <a href="https://static.igem.org/mediawiki/2015/d/d1/Aalto-Helsinki_submittedparts_colony_pcr_validation.png"><img src="https://static.igem.org/mediawiki/2015/d/d1/Aalto-Helsinki_submittedparts_colony_pcr_validation.png" style="width:102px;"/></a>
 +
  <figcaption><b><center>Figure 3.</b> Propane 1 colony PCR with primers P001 &amp; VR</center></figcaption>
 
</figure>
 
</figure>
  

Revision as of 20:16, 17 September 2015

Propane 1 codes three of the ten enzymes necessary to produce propane in Escherichia coli.

Figure 1. Propane 1

Propane 1 includes 1)acyl-CoA thioester hydrolase (YciA) from Haemophilus influenzae, which catalyzes the reaction: acyl-CoA + H2O <-> CoA + a carboxylate, 2) Carboxylic acid reductase (CAR) from Mycobacterium marinum (strain ATCC BAA-535 / M), which catalyzes the reaction: a carboxylate + reduced acceptor <-> an aldehyde + acceptor + H2O, and 3) maturation factor phosphopantetheinyl transferase (sfp) from Bacillus subtilis, which activates CAR by catalyzing the transfer of a prosthetic phosphopanteine group to CAR.

Propane 1 can be used as a part to produce propane in E. coli. The propane production pathway is shown below, and the enzymes included in Propane 1 are highlighted in blue.

All coding regions are assembled as an operon after a T7 promoter and and RBS from common cloning vectors (such as pCDF-Duet1 by Novagen). Terminator sequence after the last coding sequence, CAR is also derived from common cloning vectors (e.g. pCDF-Duet1 by Novagen uses this terminator).

All coding sequences have been codon optimized twice for E. coli. First with Thermo Fisher's codon optimizer and then with IDT's codon optimization software.

The plasmid has been assembled from IDT's gBlocks with NEBuilder assembly, similar to Gibson Assembly.

Validation: We restricted our Propane 1 and ran the insert on an agarose gel. From the picture we can tell that the insert's size is correct. The result can be seen in Figure 2.
Additionally, we did a colony PCR with VR and our primer P001. The VR primer attaches to our plasmid's backbone while P001 anneals with the very beginning of our construct. With this colony PCR we were able to show that the insert is present and it is indeed in the pSB1C3 backbone. See figure 3 for results, where the product in wells 1, and 5-10 is of the right size.

The colony which produced the product seen in figure 3, well 10 was sent to the registry.

Click here to download the full sequence of Propane 1 in pSB1C3 backbone.

Click the images to enlarge them.

Figure 2. Restriction digestion
Figure 3. Propane 1 colony PCR with primers P001 & VR

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2902
    Illegal BamHI site found at 573
    Illegal BamHI site found at 1271
    Illegal BamHI site found at 3791
    Illegal BamHI site found at 3929
    Illegal XhoI site found at 3690
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1379
    Illegal NgoMIV site found at 3608
  • 1000
    COMPATIBLE WITH RFC[1000]