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. 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.
Experimental Design
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. 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.
Sequencing Results
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 694
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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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