IGEM:MIT/2009/pycA Synthesis Plan: Difference between revisions
Shuo Cory Li (talk | contribs) |
Shuo Cory Li (talk | contribs) |
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==Mr. Gene Optimized== | ==Mr. Gene Optimized== | ||
Pushed through GeneArt's optimization server | Pushed through GeneArt's optimization server | ||
===Parameters=== | |||
Avoid | |||
* EcoRI GAATTC | |||
* Eukaria: (consensus) Splice-Donor (01) | |||
* Eukaria: (consensus) Splice-Donor (02) | |||
* Eukaria: poly(A)-site (01) | |||
* Eukaria: poly(A)-site (02) | |||
* NotI GCGGCCGC | |||
* PacI TTAATTAA | |||
* Prokaria: (consensus) TATA-Box | |||
* Prokaria: -35 Box (01) | |||
* Prokaria: -35 Box (02) | |||
* Prokaria: RBS-Entry (01) | |||
* Prokaria: RBS-Entry (02) | |||
* PstI CTGCAG | |||
* SpeI ACTAGT | |||
* XbaI TCTAGA | |||
* XhoI CTCGAG | |||
* Yeast: poly(A) UE (01) | |||
* Yeast: poly(A) UE (02) | |||
* Yeast: Splice Donor (01) | |||
* Yeast: Splice Donor (02) | |||
===Output=== | |||
<pre> | <pre> |
Revision as of 09:31, 16 June 2009
Synthesis Plan
Plan for synthesis of the pycA gene and expression in yeast. Please email back if anyone notices any major issue before we send out the order for the synthesis.
Construct
The plan is to synthesis the entire pcyA gene, flanked by the biobrick prefix and suffix so that we can insert the gene into our expression vector as well as directly deposit the part into the registry
[Biobrick Prefix] + [Kozak/RE] + [MTS] + [pcyA] + [RE] + [Biobrick Suffix]
Biobrick Prefix - gaattcgcggccgcttctag
Biobrick Suffix - tactagtagcggccgctgcag
MTS - atgcaacgctccatttttgcgaggttc - (Met - Gln - Arg - Ser - Ile - Phe - Ala - Arg - Phe)
pcyA - See below
Complete Sequence - Image on the right
This will be ligated into the plasmid YCp22FL1 via XhoI + PacI double digestion (Buffer 4 + BSA). This will not only create our desired sticky ends, but also remove the biobrick bookends. See the annotated sequence on the right for a clearer picture.
Vector
YCp22FL1 (Genbank .GB file)
YCp22FL1 is cut at the Kozak sequence and in the middle of the Firefly luciferase via XhoI and PacI. pcyA is then ligated into this region. We transform this into yeast and then pray.
Non-MTS Construct
Because we also want a version of pcyA without the MTS region, the plan is to design primers that allow for this.
Forward (5'-3')
buf kozak met pcya gcg ctcgagaacat atg gctgttactgatttgtctttgactaattct
Stats
- Melting: 55.7 C
- Worst Hairpin: -1.47 kcal/mol
- Worst Self-Dimer: -9.96 kcal/mol
Reverse (5'-3', forward direction, needs to be reverse complimented)
pcya PacI buf atgtctcaagttttgtttgatgttattcaataataa ttaattaa gcg
Stats
- Melting: 55.2 C
- Worst Hairpin: -0.46 kcal/mol
- Worst Self-Dimer: -13.61 kcal/mol
Notes
Of course, the issue is that these primers are far from ideal. The pacI site (ttaattaa) allows for very strong homodimers. pacI was chosen however because the normal cloning sites, EcoRI and XbaI, are both found in the actual gene itself, and pacI was one of the few available unique restriction sites that was easy to use / readily available.
pcyA Sequence
The optimized and unoptimized pcyA sequence. Original sequence is from the registry, part BBa_I15009
Unoptimized
The unoptimized sequence contains 4 instances of a codon that has the absolute lowest expression level in yeast
>BBa_I15009 Part-only sequence (750 bp) atggccgtcactgatttaagtttgaccaattcttccctgatgcctacgttgaacccgatgattcaacagttggccctggcgatcgccgctagttggcaaa gtttacccctcaagccctatcaattgccggaggatttgggctacgtagaaggccgcctggaaggggaaaagttagtgattgaaaatcggtgctaccaaac gccccagtttcgcaaaatgcatttggagttggccaaggtgggcaaagggttggatattctccactgtgtaatgtttcctgagcctttatacggtctacct ttgtttggctgtgacattgtggccggccccggtggagtaagtgcggctattgcggatctatcccccacccaaagcgatcgccaattgcccgcagcgtacc aaaaatcattggcagagctaggccagccagaatttgagcaacaacgggaattgcccccctggggagaaatattttctgaatattgtttattcatccgtcc cagcaatgtcactgaagaagaaagatttgtacaaagggtagtggactttttgcaaattcattgtcaccaatccatcgttgccgaacccttgtctgaagct caaactttggagcaccgtcaggggcaaattcattactgccaacaacaacagaaaaatgataaaacccgtcgggtactggaaaaagcttttggggaagctt gggcggaacggtatatgagccaagtcttatttgatgttatccaataataa
Raw Optimized
Agreed to not use
>Optimized BBa_I15009 (750 bp) ATGGCTGTTACTGATTTGTCTTTGACTAATTCTTCTTTGATGCCAACTTTGAATCCAATG ATTCAACAATTGGCTTTGGCTATTGCTGCTTCTTGGCAATCTTTGCCATTGAAACCATAT CAATTGCCAGAAGATTTGGGTTATGTTGAAGGTAGATTGGAAGGTGAAAAATTGGTTATT GAAAATAGATGTTATCAAACTCCACAATTTAGAAAAATGCATTTGGAATTGGCTAAAGTT GGTAAAGGTTTGGATATTTTGCATTGTGTTATGTTTCCAGAACCATTGTATGGTTTGCCA TTGTTTGGTTGTGATATTGTTGCTGGTCCAGGTGGTGTTTCTGCTGCTATTGCTGATTTG TCTCCAACTCAATCTGATAGACAATTGCCAGCTGCTTATCAAAAATCTTTGGCTGAATTG GGTCAACCAGAATTTGAACAACAAAGAGAATTGCCACCATGGGGTGAAATTTTTTCTGAA TATTGTTTGTTTATTAGACCATCTAATGTTACTGAAGAAGAAAGATTTGTTCAAAGAGTT GTTGATTTTTTGCAAATTCATTGTCATCAATCTATTGTTGCTGAACCATTGTCTGAAGCT CAAACTTTGGAACATAGACAAGGTCAAATTCATTATTGTCAACAACAACAAAAAAATGAT AAAACTAGAAGAGTTTTGGAAAAAGCTTTTGGTGAAGCTTGGGCTGAAAGATATATGTCT CAAGTTTTGTTTGATGTTATTCAATAATAA
Mr. Gene Optimized
Pushed through GeneArt's optimization server
Parameters
Avoid
- EcoRI GAATTC
- Eukaria: (consensus) Splice-Donor (01)
- Eukaria: (consensus) Splice-Donor (02)
- Eukaria: poly(A)-site (01)
- Eukaria: poly(A)-site (02)
- NotI GCGGCCGC
- PacI TTAATTAA
- Prokaria: (consensus) TATA-Box
- Prokaria: -35 Box (01)
- Prokaria: -35 Box (02)
- Prokaria: RBS-Entry (01)
- Prokaria: RBS-Entry (02)
- PstI CTGCAG
- SpeI ACTAGT
- XbaI TCTAGA
- XhoI CTCGAG
- Yeast: poly(A) UE (01)
- Yeast: poly(A) UE (02)
- Yeast: Splice Donor (01)
- Yeast: Splice Donor (02)
Output
>Mr. Gene Optimized BBa_I15009 (750 bp) ATGGCCGTTACCGATTTGAGTTTGACCAATTCCTCCTTGATGCCAACCTTAAACCCTATGATTCAACAATTGGCTTTGGCTATTGCTGCTTCCTGGCAATCTTTGCCTTTG AAACCATATCAATTGCCTGAAGATTTGGGTTATGTCGAAGGTAGATTAGAAGGTGAAAAATTGGTTATCGAAAACAGATGCTATCAAACCCCACAATTCAGAAAAATGCAC TTGGAATTGGCTAAAGTCGGTAAAGGTTTAGACATCTTACACTGTGTCATGTTCCCTGAACCATTGTATGGTTTACCATTATTCGGTTGTGACATCGTTGCTGGTCCTGGT GGTGTCTCTGCTGCCATTGCCGATTTGTCTCCAACACAATCCGATAGACAATTGCCTGCTGCCTATCAAAAATCCTTGGCCGAATTGGGTCAACCAGAATTTGAACAACAA AGAGAATTGCCTCCTTGGGGTGAAATTTTCTCCGAATATTGTTTGTTCATTAGACCATCCAACGTCACCGAAGAAGAAAGATTCGTCCAAAGAGTTGTCGACTTCTTACAA ATCCACTGCCACCAATCCATCGTAGCCGAACCATTATCCGAAGCTCAAACATTGGAACACAGACAAGGTCAAATCCATTATTGCCAACAACAACAAAAAAACGACAAGACT AGAAGAGTTTTGGAAAAGGCTTTCGGTGAAGCTTGGGCCGAAAGATATATGTCCCAAGTTTTATTCGACGTCATTCAATGATGA
Cost / Logistics
We have a 2,500 bp limit for the special rates of $0.2/bp.
The construct itself is 843bp. At 0.2$/bp, it is roughly 170 dollars. Plus $35 for shipping, total ends up at $204
Turnaround is ~ 15 days.
This also leaves us with roughly 1657bp left for additional synthesis at the special iGEM price.