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]

Complete Construct Sequence
(Genbank .GB File)

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

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

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.