IGEM:MIT/2009/pycA Synthesis Plan

=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

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

Codon Usage Analysis >BBa_I15009 Part-only sequence (750 bp) atggccgtcactgatttaagtttgaccaattcttccctgatgcctacgttgaacccgatgattcaacagttggccctggcgatcgccgctagttggcaaa gtttacccctcaagccctatcaattgccggaggatttgggctacgtagaaggccgcctggaaggggaaaagttagtgattgaaaatcggtgctaccaaac gccccagtttcgcaaaatgcatttggagttggccaaggtgggcaaagggttggatattctccactgtgtaatgtttcctgagcctttatacggtctacct ttgtttggctgtgacattgtggccggccccggtggagtaagtgcggctattgcggatctatcccccacccaaagcgatcgccaattgcccgcagcgtacc aaaaatcattggcagagctaggccagccagaatttgagcaacaacgggaattgcccccctggggagaaatattttctgaatattgtttattcatccgtcc cagcaatgtcactgaagaagaaagatttgtacaaagggtagtggactttttgcaaattcattgtcaccaatccatcgttgccgaacccttgtctgaagct caaactttggagcaccgtcaggggcaaattcattactgccaacaacaacagaaaaatgataaaacccgtcgggtactggaaaaagcttttggggaagctt gggcggaacggtatatgagccaagtcttatttgatgttatccaataataa

Raw Optimized
Codon Usage Analysis

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.