Difference between revisions of "Part:BBa K2253002"
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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. | 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. | ||
| + | <div align="center"> | ||
| + | https://static.igem.org/mediawiki/parts/thumb/3/34/Blue_cp_transformation.JPG/247px-Blue_cp_transformation.JPG.png | ||
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| − | <b>Figure 1.</b> Transformation plate of the K60800 vector and codon optimized blue chromoprotein ligation reaction. | + | <b> Figure 1.</b> Transformation plate of the K60800 vector and codon optimized blue chromoprotein ligation reaction. |
===Sequencing Results=== | ===Sequencing Results=== | ||
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Revision as of 01:36, 1 November 2017
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. After purifying this ligation, a transformation was performed, which yielded in one phentotypically blue colony, shown below.
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.