Difference between revisions of "Part:BBa K2253002"
 
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This is the original blue chromoprotein sequence (BBa_K592009) that has been codon optimized. The original part has been made a basic part with the promoter and RBS sequence, BBa_K608002 in the pSB1C3 BioBrick backbone. Codon optimization allows it to function more efficiently in E.coli, therefore creating a lower metabolic burden on the cells that it is encoded in. When this sequence is put into a vector and transformed into a plasmid it shows a blue phenotype. Codon optimization also makes this part more stable, as the color is expected to be maintained for a longer period of time than the original, making it a good candidate to use as a biosensor for things like toxins, such as lead.  
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This is the original blue chromoprotein sequence (<a href="https://parts.igem.org/Part:BBa_K592009"> BBa_K592009 </a>) that has been codon-optimized. The codon-optimized blue chromoprotein containing the strong promoter and RBS sequence from BBa_K608002 has been made a basic part. Normally, the original BBa_K592009 blue chromoprotein part is unstable due to numerous mutations in its sequence, caused by the high metabolic burden expressed on the cell<sup>[1]</sup>. Codon optimization involves exchanging certain codons for ones that have known to be more translationally efficient in a certain species while retaining the same sequence of amino acids. It can potentially improve the originally unstable blue chromoprotein to function more efficiently in <i>E. coli</i>, therefore creating a lower metabolic burden on the cells in which it is expressed in. The blue color from the codon-optimized blue chromoprotein is expected to be maintained for a longer period of time than the original part, making it a good candidate as a biosensor for things like toxins, such as lead.  
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===Experimental Design===
 
===Experimental Design===
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To attempt to improve the stability of the original blue chromoprotein, a digestion was performed on the K608002 vector and the codon optimized blue chromoprotein, which was ordered as a G-block from the Integrated DNA Technologies (IDT) website. Upon digesting each of these parts and conducting a gel extraction, a ligation reaction was used to ligate the gene of interest (codon optimized blue chromoprotein) into the K608002 vector. After purifying this ligation, a transformation was performed, which yielded in one phentotypically blue colony, shown below.  
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To attempt to improve the stability of the original blue chromoprotein, a digestion was performed on the BBa_K608002 vector and the codon-optimized blue chromoprotein, which was ordered as a gBlock from the Integrated DNA Technologies (IDT) website. Upon digestion of these parts and conducting gel extraction, a ligation reaction was used to join our codon-optimized blue chromoprotein part with the BBa_K608002 vector. Following the purification of the ligation, <i>E. coli</i> transformation was done via electroporation which yielded one phenotypically blue colony, shown below in <b>Figure 1.</b> This colony was then inoculated into liquid media to make an overnight culture, shown in <b>Figure 2,</b> which maintained a blue phenotype.  
  
 
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<b> Figure 1.</b> Transformation plate of the K60800 vector and codon optimized blue chromoprotein ligation reaction.
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<b> Figure 1.</b> Transformation plate of the K608002 vector and codon optimized blue chromoprotein ligation reaction.
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https://static.igem.org/mediawiki/parts/thumb/7/74/Codon_op_blue_cp.png/162px-Codon_op_blue_cp.png
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<b> Figure 2.</b> Overnight culture made from inoculated colony on transformation plate in Figure 1. Liquid culture shows blue phenotype.
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===Sequencing Results===
 
===Sequencing Results===
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In comparison to the original blue chromoprotein sequence in BBa_K592009, our sequenced codon-optimized blue chromoprotein contained fewer mutations.
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<partinfo>BBa_K2253002 SequenceAndFeatures</partinfo>
 
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Through the codon optimization of this part, in comparison to the BBa_K592009 original chromoprotein, the sequenced codon optimized blue chromoprotein shows minimal mutations.
 
  
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===References===
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#Sleight et al. Journal of Biological Engineering 4(12) 1-20 (2010)
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2253002 parameters</partinfo>
 
<partinfo>BBa_K2253002 parameters</partinfo>
 
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Latest revision as of 01:40, 2 November 2017


Optimized Blue Chromoprotein

This is the original blue chromoprotein sequence ( BBa_K592009 ) that has been codon-optimized. The codon-optimized blue chromoprotein containing the strong promoter and RBS sequence from BBa_K608002 has been made a basic part. Normally, the original BBa_K592009 blue chromoprotein part is unstable due to numerous mutations in its sequence, caused by the high metabolic burden expressed on the cell[1]. Codon optimization involves exchanging certain codons for ones that have known to be more translationally efficient in a certain species while retaining the same sequence of amino acids. It can potentially improve the originally unstable blue chromoprotein to function more efficiently in E. coli, therefore creating a lower metabolic burden on the cells in which it is expressed in. The blue color from the codon-optimized blue chromoprotein is expected to be maintained for a longer period of time than the original part, making it a good candidate as a biosensor for things like toxins, such as lead.

Experimental Design

To attempt to improve the stability of the original blue chromoprotein, a digestion was performed on the BBa_K608002 vector and the codon-optimized blue chromoprotein, which was ordered as a gBlock from the Integrated DNA Technologies (IDT) website. Upon digestion of these parts and conducting gel extraction, a ligation reaction was used to join our codon-optimized blue chromoprotein part with the BBa_K608002 vector. Following the purification of the ligation, E. coli transformation was done via electroporation which yielded one phenotypically blue colony, shown below in Figure 1. This colony was then inoculated into liquid media to make an overnight culture, shown in Figure 2, which maintained a blue phenotype.

247px-Blue_cp_transformation.JPG.png

Figure 1. Transformation plate of the K608002 vector and codon optimized blue chromoprotein ligation reaction.

162px-Codon_op_blue_cp.png

Figure 2. Overnight culture made from inoculated colony on transformation plate in Figure 1. Liquid culture shows blue phenotype.

Sequencing Results

In comparison to the original blue chromoprotein sequence in BBa_K592009, our sequenced codon-optimized blue chromoprotein contained fewer mutations.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 694
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

  1. Sleight et al. Journal of Biological Engineering 4(12) 1-20 (2010)