Difference between revisions of "Part:BBa K3634006:Design"
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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." | 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: | 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(25): Removed by a27g, GAA (Glu) to GAg (Glu). |
| − | EcoR1(517): Removed by a519g, 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) | + | EcoR1(1603): Removed by a1605g, GAA (Glu) to GAg (Glu). |
| − | PstI(1163): Removed by g1161c, CTG (Leu) to CTc (Leu) | + | PstI(1163): Removed by g1161c, CTG (Leu) to CTc (Leu). |
| − | SapI(2185): Removed by g2190c, 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. | This allowed for the part to be used at RFC[10] and RFC[1000] standard with codon optimisation. | ||
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===Source=== | ===Source=== | ||
Revision as of 10:51, 5 August 2020
ccaS (Codon Optimised for E.coli)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2078
Illegal BamHI site found at 1417 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1449
- 1000COMPATIBLE WITH RFC[1000]
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.