Part:BBa_K4011016
pTEF1-SaACS2-tADH1
TEF1-SaACS2-tADH1 is an expression cassette in S. cerevisiae capable of expressing SaACS2, which will synthesize acetyl-CoA from acetate. Our final goal is to synthesize ethyl-acetate in yeast. pTEF1 BBa_K319003, SaACS2 BBa_K4011007, and tADH1 BBa_K4012022 were used to construct pTEF1-SaACS2-tADH1 using Golden Gate assembly. This part was used to construct pTEF1-SaACS2-tADH1-pRPL8B-AeAT9-tSSA1 BBa_K4011018.
Usage and Biology
SaACS2 is an acetyl-CoA synthase from Salmonella enterica. Its natural function is to convert acetate into acetyl-CoA under anaerobic conditions. Its enzymatic rate is around 50 times that of ACS1 found in S. cerevisiae. pTEF1 is a relatively strong promoter and tADH1 is a medium strength terminator both characterized by Lee et al in 2021.
Source
pTEF1 and tADH1 all come from S. cerevisiae, while SaACS2 comes from Salmonella enterica.
Design Considerations
1. All codons were optimized for S. cerevisiae based on S. cerevisiae codon bias.
2. BsaI restriction sites were added to the three basic parts used to construct pTEF1-SaACS2-tADH1 for Golden Gate assembly.
3. BsmB1 sites flank the composite part for further Golden Gate assembly construction.
Characterization
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 2426
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1987
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2412
Illegal BsaI.rc site found at 205
Illegal BsaI.rc site found at 2662
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