Part:BBa_K2253002

Optimized Blue Chromoprotein

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. 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 that it is encoded in. Codon optimization can also make this part more stable, as the color is expected to be maintained for a longer period of time than the original part, making it a good candidate to use as a biosensor for things like toxins, such as lead. It is important to note that when this sequence is put into a vector and transformed into a plasmid, it shows a blue phenotype.

Experimental Design

To attempt to improve the stability of the original blue chromoprotein, a digestion was performed on the BBa_K608002 vector (within the PSB1C3 backbone) 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. Following the purification of the ligation, a transformation was done via electroporation which yielded one phentotypically blue colony, shown below in Figure 1. This colony was then inoculated into liquid media to make an overnight culture, shown in Figure 2.

Figure 1. Transformation plate of the K60800 vector and codon optimized blue chromoprotein ligation reaction.

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

Sequencing Results

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]

Through the codon optimization of this part, in comparison to the BBa_K592009 original chromoprotein sequence, the sequenced codon optimized blue chromoprotein in the BBa_K608002 vector in the pSB1C3 BioBrick backbone shows minimal mutations.

References

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