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Team:Frankfurt/Story
From 2012.igem.org
Contents |
DNA constructs for realisation of our idea
Mevalonate Pathway Overexpression
The vector for improving the MVA pathway flow contains three genes: A trunctated version of the HMG-CoA-Reductase, the native gene of the Farnesylpyrophosphate Synthase (ERG20) and the GGPP Synthase gene from Sulfolobius acileratius. The sources of the physical DNA were as following:
- HMG-CoA-Reductase: The sequence was obtained from the Saccharomyces Genome Database and modified. This version was send to a company for de novo synthesis.
- ERG20: The gene was obtained from genomic DNA of the strain CEN.PK2. By using primers which contained overlaps with the pre- and suffix sequences it was amplified by PCR.
- GGPP-Synthase : The sequence was obtained from the Pubmed Database and modified. This version was send to a company for de novo synthesis.
construction of the insert:
pHXT7 HMG-CoA tHXT7 pPFK1 ERG20 tPFK2 pPGK1 GGPPS tCYC1
plasmid backbone with URA3
Steviol Production
The vector for steviol production also contains three genes: The bifunctional cyclase from Gibberella fujikuroi (CPS/KS), the ent-kaurene oxidase from Gibberella fujikuroi (KO) and the ent-kaurenoic acid hydroxylase from Stevia rebaudiana (KAH). The sources of the physical DNA were as following:
- bifunctional cyclase: The sequence was obtained from the Pubmed Database and modified. This version was send to a company for de novo synthesis.
- ent-kaurene oxidase: The sequence was obtained from the Pubmed Database and modified. This version was send to a company for de novo synthesis (4 x 500 bp for Gibson Assembly).
- ent-kaurenoic acid hydroxylase: The sequence was obtained from the Pubmed Database and modified. This version was send to a company for de novo synthesis (4 x 500 bp for Gibson Assembly).
construction of the insert:
pHXT7 CPS/KS tTAL1 pTPI1 KO tPDC1 pPGI1 KAH tCYC1
plasmid backbone with HIS3
DNA sources
| Templates | Amplified DNA Fragments |
|---|---|
| synthesized sequence of HMG-CoA | HMG-CoA |
| synthesized sequence of GGPPS | GGPPS |
| chromosomal DNA of CEN.PK2-1C | ERG20 |
| synthesized sequence of Cps/Ks | CPS/KS |
| synthesized sequence of KO | ent-kaurene oxidase |
| synthesized sequence of KAH | ent-kaurenoic acid hydroxylase |
| pUD8e (yeast shuttle plasmid) | pPFK1, pTPI1, tHXT7, tTAL1 |
| pUD22e (yeast shuttle plasmid) | pPGK1, pPGI1, tPFK2, tPDC1 |
Sequence Editing
The most of our genes were synthesized, because they contained several biobrick restriction sites. The relevant codons were replaced by different codons which encode for the same amino acid. Only ERG20 has no biobrick restriction sites.
- HMG-CoA
synthesized sequence
5`: 45 bp overlap to pHXT7 and the prefix
3`: the suffix and 33 bp overlap to tHXT7
- ERG20
gene from chromosomal DNA of CEN.PK2-1C
5`: 16 bp overlap to pPFK1 and the prefix (added via primer)
3`: the suffix and 14 bp overlap to tPFK2 (added via primer)
- GGPPS
synthesized sequence
5`: 18 bp overlap to pPGK1 and the prefix
3`: the suffix and 45 bp overlap to tCYC1
- CPS/KS
synthesized sequence
5': 45 bp overlap to PHXT7 and the prefix
3': the suffix and 17 bp overlap to tTAL1
- KO
synthesized sequence (4 x 500 bp for Gibson Assembly)
5': 15 bp overlap to pTPI1 and the prefix
3': the suffix and 16 bp overlap to tPDC1
- KAH
synthesized sequence (4 x 500 bp for Gibson Assembly)
5': 15 bp overlap to pPGI1 and the prefix
3': the suffix and 45 bp overlap to tCYC1
- promoters and terminators
They were amplified from plasmids via PCR with designed primers.
The primer contain about 20 to 30 bp of the promoter or terminator sequence for PCR and about 30 bp overlap to the neighbouring sequence for homologue recombination.
