DIY: Gene synthesis codon optimization

Do not use free codon optimization algorithm provided by some
"leading" companies, such as GeneArt, Genscript,  IDT etc. A
company has recently synthesized ~ 100 genes using GeneArt software, or
sequences provided by Genscript and IDT, 90% of the synthetic genes
could not be expressed in E coli, or expressed at extremely low
level (compared to wt).

Save money to do your own optimization for expression of your gene in
E coli:

1. Analyse wild type DNA sequence using

http://54.209.1.228/rare_codon.html

 

2. Change  rare codons 
Ile: ATA
Leu: CTA
Pro: CCC
Arg: AGG, AGA, CGG, and CGA
based on this
E coli codon usage table:

http://www.my-whiteboard.com/e-coli-codon-usage-table-2/

For example:

L:
cta –> CTG

I:
ata –> ATC or ATT

R:
aga –> CGC or CGT
cgg –> CGC or CGT

cga–> CGC or CGT
agg –> CGC or CGT

P:
ccc –> CCG

T:

ACA–>ACC,  ACG

G:

GGA–>GGT, GGC
GGG–>GGT, GGC

3. Change second amino acid to A (gct, gca), K (aaa) or S (agc, tcc, tct)

4. Change G and C to A and T at the 5'-end.
A high GC content in the 5'-end of the gene of interest –> formation of secondary structure in the mRNA —> Inefficient translation —> low expression

5.  Remove cis-acting DNA sequences such as internal TATA-boxes, chi-sites, and ribosomal entry sites; AT-rich or GC-rich sequence stretches; repeat sequences; and RNA secondary structures. Remove internal Shine-Dalgarno sequences such as AAGGAG(nnnnn)ATG, GAAGGAGA(nnnnn)ATG, AAGGAGG(nnnnn)ATG, AAGGAGGT(nnnnn)ATG, GGAG, GAGG, and AGGA.

For example, you should change ATAATA to ATCATC; change AGAAGA to CGCCGT; ATAAGA to ATCCGT; AGAATA to CGCATT; ATAAGG to ATTCGT; AGAAGG to CGTCGC

 

6. Add two stop codons TAATAA

7. Append another strong terminator to the end of your DNA sequence

 

References:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC139613/

December 22, 2012 at 3:59 pm

1 Comment »

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    December 10, 2014 @ 8:08 am

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