IGEM:MIT/2009/pycA Synthesis Plan: Difference between revisions
Shuo Cory Li (talk | contribs) |
Shuo Cory Li (talk | contribs) |
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<pre> | <pre> | ||
>pycA Optimized (DNA2.0) | >pycA Optimized (DNA2.0) | ||
ATGGCTGTGACTGATTTGTCATTGACAAACAGTTCTTTGATGCCAACTCTGAACCCAATGATACAACAGCTTGCACTGGCTATTGCTGCTAGTTGGCAATCTCT | |||
ACCTCTTAAACCATACCAATTACCAGAAGATCTGGGTTACGTGGAGGGTAGACTTGAGGGTGAGAAGCTGGTGATTGAGAATAGATGCTATCAAACTCCACAGT | |||
TCAGAAAGATGCACTTGGAGTTAGCTAAAGTTGGTAAAGGGTTAGACATCTTACATTGCGTTATGTTCCCTGAACCTTTGTACGGATTGCCATTGTTTGGTTGT | |||
GATATTGTAGCAGGACCTGGTGGTGTATCCGCTGCCATTGCAGATCTTTCACCAACTCAGTCTGATCGTCAACTACCAGCTGCCTACCAAAAGTCTTTGGCAGA | |||
ATTAGGACAACCAGAGTTCGAACAACAAAGAGAACTGCCACCTTGGGGCGAAATCTTTTCTGAATACTGTTTGTTCATCAGACCATCCAATGTTACCGAGGAAG | |||
AAAGGTTCGTCCAAAGAGTCGTTGATTTCTTGCAAATACATTGTCATCAATCTATTGTTGCCGAACCTTTATCTGAAGCACAAACACTAGAACATAGACAGGGC | |||
CAAATACACTATTGTCAACAACAGCAGAAAAACGATAAGACAAGAAGAGTACTAGAAAAGGCATTTGGGGAGGCTTGGGCAGAAAGATACATGTCACAAGTCCT | |||
ATTTGACGTTATCCAGTAATAA | |||
</pre> | </pre> | ||
==Raw Output== | ==Raw Output== |
Latest revision as of 12:40, 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
Server was asked to 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
DNA 2.0 Quote Optimized
Received back this quote from DNA 2.0
Sequence
>pycA Optimized (DNA2.0) ATGGCTGTGACTGATTTGTCATTGACAAACAGTTCTTTGATGCCAACTCTGAACCCAATGATACAACAGCTTGCACTGGCTATTGCTGCTAGTTGGCAATCTCT ACCTCTTAAACCATACCAATTACCAGAAGATCTGGGTTACGTGGAGGGTAGACTTGAGGGTGAGAAGCTGGTGATTGAGAATAGATGCTATCAAACTCCACAGT TCAGAAAGATGCACTTGGAGTTAGCTAAAGTTGGTAAAGGGTTAGACATCTTACATTGCGTTATGTTCCCTGAACCTTTGTACGGATTGCCATTGTTTGGTTGT GATATTGTAGCAGGACCTGGTGGTGTATCCGCTGCCATTGCAGATCTTTCACCAACTCAGTCTGATCGTCAACTACCAGCTGCCTACCAAAAGTCTTTGGCAGA ATTAGGACAACCAGAGTTCGAACAACAAAGAGAACTGCCACCTTGGGGCGAAATCTTTTCTGAATACTGTTTGTTCATCAGACCATCCAATGTTACCGAGGAAG AAAGGTTCGTCCAAAGAGTCGTTGATTTCTTGCAAATACATTGTCATCAATCTATTGTTGCCGAACCTTTATCTGAAGCACAAACACTAGAACATAGACAGGGC CAAATACACTATTGTCAACAACAGCAGAAAAACGATAAGACAAGAAGAGTACTAGAAAAGGCATTTGGGGAGGCTTGGGCAGAAAGATACATGTCACAAGTCCT ATTTGACGTTATCCAGTAATAA
Raw Output
>Li_NoName_061609_opt GCGGAATTCGCGGCCGCTTCTAGAGCTCGAGAACATATGGCTGTGACTGATTTGTCATTGACAAACAGTTCTTTGATGCCAACTCTGAACCCAATGATACAACA GCTTGCACTGGCTATTGCTGCTAGTTGGCAATCTCTACCTCTTAAACCATACCAATTACCAGAAGATCTGGGTTACGTGGAGGGTAGACTTGAGGGTGAGAAGC TGGTGATTGAGAATAGATGCTATCAAACTCCACAGTTCAGAAAGATGCACTTGGAGTTAGCTAAAGTTGGTAAAGGGTTAGACATCTTACATTGCGTTATGTTC CCTGAACCTTTGTACGGATTGCCATTGTTTGGTTGTGATATTGTAGCAGGACCTGGTGGTGTATCCGCTGCCATTGCAGATCTTTCACCAACTCAGTCTGATCG TCAACTACCAGCTGCCTACCAAAAGTCTTTGGCAGAATTAGGACAACCAGAGTTCGAACAACAAAGAGAACTGCCACCTTGGGGCGAAATCTTTTCTGAATACT GTTTGTTCATCAGACCATCCAATGTTACCGAGGAAGAAAGGTTCGTCCAAAGAGTCGTTGATTTCTTGCAAATACATTGTCATCAATCTATTGTTGCCGAACCT TTATCTGAAGCACAAACACTAGAACATAGACAGGGCCAAATACACTATTGTCAACAACAGCAGAAAAACGATAAGACAAGAAGAGTACTAGAAAAGGCATTTGG GGAGGCTTGGGCAGAAAGATACATGTCACAAGTCCTATTTGACGTTATCCAGTAATAATTAATTAATACTAGTAGCGGCCGCTGCAGGCG >5RE GCGGAATTCGCGGCCGCTTCTAGAGCTCGAGAACAT >Li_NoName_061609 ATGGCTGTGACTGATTTGTCATTGACAAACAGTTCTTTGATGCCAACTCTGAACCCAATGATACAACAGCTTGCACTGGCTATTGCTGCTAGTTGGCAATCTCT ACCTCTTAAACCATACCAATTACCAGAAGATCTGGGTTACGTGGAGGGTAGACTTGAGGGTGAGAAGCTGGTGATTGAGAATAGATGCTATCAAACTCCACAGT TCAGAAAGATGCACTTGGAGTTAGCTAAAGTTGGTAAAGGGTTAGACATCTTACATTGCGTTATGTTCCCTGAACCTTTGTACGGATTGCCATTGTTTGGTTGT GATATTGTAGCAGGACCTGGTGGTGTATCCGCTGCCATTGCAGATCTTTCACCAACTCAGTCTGATCGTCAACTACCAGCTGCCTACCAAAAGTCTTTGGCAGA ATTAGGACAACCAGAGTTCGAACAACAAAGAGAACTGCCACCTTGGGGCGAAATCTTTTCTGAATACTGTTTGTTCATCAGACCATCCAATGTTACCGAGGAAG AAAGGTTCGTCCAAAGAGTCGTTGATTTCTTGCAAATACATTGTCATCAATCTATTGTTGCCGAACCTTTATCTGAAGCACAAACACTAGAACATAGACAGGGC CAAATACACTATTGTCAACAACAGCAGAAAAACGATAAGACAAGAAGAGTACTAGAAAAGGCATTTGGGGAGGCTTGGGCAGAAAGATACATGTCACAAGTCCT ATTTGACGTTATCCAGTAATAA >3RE TTAATTAATACTAGTAGCGGCCGCTGCAGGCG Translation Map Li_NoName_061609 1 ATGGCTGTGACTGATTTGTCATTGACAAACAGTTCTTTGATGCCAACTCTGAACCCAATG 1 M A V T D L S L T N S S L M P T L N P M 61 ATACAACAGCTTGCACTGGCTATTGCTGCTAGTTGGCAATCTCTACCTCTTAAACCATAC 21 I Q Q L A L A I A A S W Q S L P L K P Y 121 CAATTACCAGAAGATCTGGGTTACGTGGAGGGTAGACTTGAGGGTGAGAAGCTGGTGATT 41 Q L P E D L G Y V E G R L E G E K L V I 181 GAGAATAGATGCTATCAAACTCCACAGTTCAGAAAGATGCACTTGGAGTTAGCTAAAGTT 61 E N R C Y Q T P Q F R K M H L E L A K V 241 GGTAAAGGGTTAGACATCTTACATTGCGTTATGTTCCCTGAACCTTTGTACGGATTGCCA 81 G K G L D I L H C V M F P E P L Y G L P 301 TTGTTTGGTTGTGATATTGTAGCAGGACCTGGTGGTGTATCCGCTGCCATTGCAGATCTT 101 L F G C D I V A G P G G V S A A I A D L 361 TCACCAACTCAGTCTGATCGTCAACTACCAGCTGCCTACCAAAAGTCTTTGGCAGAATTA 121 S P T Q S D R Q L P A A Y Q K S L A E L 421 GGACAACCAGAGTTCGAACAACAAAGAGAACTGCCACCTTGGGGCGAAATCTTTTCTGAA 141 G Q P E F E Q Q R E L P P W G E I F S E 481 TACTGTTTGTTCATCAGACCATCCAATGTTACCGAGGAAGAAAGGTTCGTCCAAAGAGTC 161 Y C L F I R P S N V T E E E R F V Q R V 541 GTTGATTTCTTGCAAATACATTGTCATCAATCTATTGTTGCCGAACCTTTATCTGAAGCA 181 V D F L Q I H C H Q S I V A E P L S E A 601 CAAACACTAGAACATAGACAGGGCCAAATACACTATTGTCAACAACAGCAGAAAAACGAT 201 Q T L E H R Q G Q I H Y C Q Q Q Q K N D 661 AAGACAAGAAGAGTACTAGAAAAGGCATTTGGGGAGGCTTGGGCAGAAAGATACATGTCA 221 K T R R V L E K A F G E A W A E R Y M S 721 CAAGTCCTATTTGACGTTATCCAGTAATAA 241 Q V L F D V I Q * * Restriction Sites Name Seq. Locations AatI AGGCCT none AccI GTMKAC 187 AflII CTTAAG none AgeI ACCGGT none AlwI GGATC none AlwNI CAGNNNCTG 358 ApaI GGGCCC none ApaLI GTGCAC none AscI GGCGCGCC none AseI ATTAAT 785, 789 AvaI CYCGRG 25 AvaII GGWCC 360 AvrII CCTAGG none BamHI GGATCC none BbsI GAAGAC none BbvI GCAGC 120(c), 378(c), 426(c), 808(c) BclI TGATCA none BglI GCCNNNNNGGC none BglII AGATCT 167, 389 BlpI GCTNAGC none BsaI GGTCTC none BsmAI GTCTC none BsmBI CGTCTC none BstEII GGTNACC none BstXI CCANNNNNNTGG none ClaI ATCGAT none DraIII CACNNNGTG none EagI CGGCCG 10, 803 EarI CTCTTC 703(c) EcoRI GAATTC 3 EcoRV GATATC none FokI GGATG 535(c) FseI GGCCGGCC none HindIII AAGCTT none KasI GGCGCC none KpnI GGTACC none MluI ACGCGT none NarI GGCGCC none NcoI CCATGG none NdeI CATATG 33 NheI GCTAGC none NotI GCGGCCGC 9, 802 NsiI ATGCAT none PacI TTAATTAA 786 PciI ACATGT 748 PmeI GTTTAAAC none PstI CTGCAG 809 PvuI CGATCG none PvuII CAGCTG 424 SacI GAGCTC 22 SacII CCGCGG none SalI GTCGAC none SapI GCTCTTC none SfiI GGCCNNNNNGGCC none SgrAI CRCCGGYG none SmaI CCCGGG none SpeI ACTAGT 795 SphI GCATGC none SspI AATATT none StuI AGGCCT none SwaI ATTTAAAT none TliI CTCGAG 25 XbaI TCTAGA 18 XhoI CTCGAG 25 XmaI CCCGGG none XmnI GAANNNNTTC none GCRun8 SSSSSSSS 8, 9, 802 T7ClassII YATCTGTW none W6 WWWWWW 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790 SpliceDonor AGGTRAG none SpliceDonor2 GGTRAGT none SpliceAcc YYYNYAGGW none SpliceAcc2 YNCAGGW none RNADestab ATTTA none A6 AAAAAA none C6 CCCCCC none G6 GGGGGG none T6 TTTTTT none ATRich AAWWAA 784, 788, 786(c) ATRich2 ATATATATA none PolyA2 AATGAA none PolyA3 AAATGGAAA none PolyA4 AATGGAAATG none SpliceDnr3 GGTAAG none SpliceAcc3 YYYNCAGRW none Codon Usage Table AmAcid Codon Number /1000 Fraction END TAA 2 8.0 1.0 END TGA 0 0.0 0.0 END TAG 0 0.0 0.0 ALA GCT 8 32.0 0.44 ALA GCA 7 28.0 0.38 ALA GCC 3 12.0 0.16 ALA GCG 0 0.0 0.0 CYS TGT 4 16.0 0.66 CYS TGC 2 8.0 0.33 ASP GAT 7 28.0 0.77 ASP GAC 2 8.0 0.22 GLU GAA 14 56.0 0.63 GLU GAG 8 32.0 0.36 PHE TTC 6 24.0 0.6 PHE TTT 4 16.0 0.4 GLY GGT 7 28.0 0.5 GLY GGA 3 12.0 0.21 GLY GGC 2 8.0 0.14 GLY GGG 2 8.0 0.14 HIS CAT 4 16.0 0.66 HIS CAC 2 8.0 0.33 ILE ATC 4 16.0 0.33 ILE ATT 5 20.0 0.41 ILE ATA 3 12.0 0.25 LYS AAG 5 20.0 0.55 LYS AAA 4 16.0 0.44 LEU TTG 10 40.0 0.33 LEU TTA 6 24.0 0.2 LEU CTA 5 20.0 0.16 LEU CTT 4 16.0 0.13 LEU CTG 5 20.0 0.16 LEU CTC 0 0.0 0.0 MET ATG 6 24.0 1.0 ASN AAC 3 12.0 0.6 ASN AAT 2 8.0 0.4 PRO CCA 11 44.0 0.64 PRO CCT 6 24.0 0.35 PRO CCC 0 0.0 0.0 PRO CCG 0 0.0 0.0 GLN CAA 17 68.0 0.70 GLN CAG 7 28.0 0.29 ARG AGA 10 40.0 0.83 ARG AGG 1 4.0 0.08 ARG CGT 1 4.0 0.08 ARG CGA 0 0.0 0.0 ARG CGC 0 0.0 0.0 ARG CGG 0 0.0 0.0 SER TCT 7 28.0 0.5 SER TCA 3 12.0 0.21 SER TCC 2 8.0 0.14 SER AGT 2 8.0 0.14 SER AGC 0 0.0 0.0 SER TCG 0 0.0 0.0 THR ACA 3 12.0 0.37 THR ACT 4 16.0 0.5 THR ACC 1 4.0 0.12 THR ACG 0 0.0 0.0 VAL GTT 6 24.0 0.4 VAL GTC 3 12.0 0.2 VAL GTA 3 12.0 0.2 VAL GTG 3 12.0 0.2 TRP TGG 3 12.0 1.0 TYR TAC 6 24.0 0.75 TYR TAT 2 8.0 0.25 GC Percentage: 43.39853300733496% Repeats greater than or equal to 10, in Li_NoName_061609_opt AGCGGCCGCT (10 bases) 802, 811 802, 811 ATTAATTAAT (10 bases) 786, 795 786, 795 TAATTAATTA (10 bases) 784, 793 784, 793
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.