Difference between revisions of "Part:BBa K3634006:Design"

Latest revision as of 10:59, 5 August 2020

ccaS (Codon Optimised for E.coli)

Design Notes

2 illegal restriction sites found in the original DNA had already been removed following extraction from Tabor's pJT122 plasmid (a1282c & a1284c [EcoR1]; t1383c [SpeI]) by Uppsala iGEM 2011. The site-directed mutagenesis g1278t was also made as part of a codon optimisation step. St Andrews iGEM 2020 then carried out a further codon optimisation step for E.coli for the whole BBa_K592001 sequence using the IDT codon optimisation tool. Following optimisation, a low complexity score (3.1) was given to the sequence indicating some complexities existed but were not anticipated to cause a problem. The description given was "one or more repeated sequences greater than 8 bases comprise 47.8% of the overall sequence. Solution: Redesign to reduce the repeats to be less than 40% of the sequence." Optimisation created 3xEcoRI, 1xPstI and 1xSapI illegal restriction sites. These were then removed by the following in silico point mutagenesis alterations: EcoR1(25): Removed by a27g, GAA (Glu) to GAg (Glu). EcoR1(517): Removed by a519g, GAA (Glu) to GAg (Glu). EcoR1(1603): Removed by a1605g, GAA (Glu) to GAg (Glu). PstI(1163): Removed by g1161c, CTG (Leu) to CTc (Leu). SapI(2185): Removed by g2190c, CTG (Leu) to CTc (Leu). This allowed for the part to be used at RFC[10] and RFC[1000] standard with codon optimisation.

Source

The system is native to Synechocystis sp. PCC6803 of which the sequence can be obtained from BBa_K592001 (Uppsala-Sweden iGEM, 2011 - initially from Tabor's pJT122 plasmid). The part sequence was then fully optimised for our chosen chassis organism, E.coli, using the IDT codon optimisation tool.

References

Uppsala-Sweden iGEM 2011 - https://parts.igem.org/Part:BBa_K592001

IDT Codon Optimisation Tool - https://eu.idtdna.com/CodonOpt

Hirose Y., Shimada T., Narikawa R., Katayama M., Ikeuchi M. 2008. Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. PNAS. 105(28): p9528-9533. DOI: 10.1073􏰃/pnas.0801826105

Tabor J.J., Levskaya A., Voigt C.A. 2011. Multichromatic control of gene expression in Escherichia coli. J Mol Biol. 405(2): p315–324. DOI: 10.1016/j.jmb.2010.10.038

Schmidl S.R., Sheth R.U., Wu A., Tabor J.J. 2014. Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems. ACS Synth Biol. 3: p820-831. DOI: 10.1021/sb500273n