How To Perform A Codon Usage Analysis

Question

[Samane]

hello, I want to use soeing PCR for two genes ( expression of two gene: B.subtilis ----to--->E.coli) then I need to use codon usage analysis for expression.can yo help me to explain how can I use codon usage analyser? as you know I have two nucleic acid strand from B.subtilis to express it to E.coli...

is there a online or other software to help me in this? and how can I use that?

thank you.

[Devon Ryan]

It's a bit unclear what the overlap PCR has to do with the codon usage. Could you supply more context surrounding what you're trying to do? My only guess would be that the overlap PCR is merging two constructs together (perhaps with an IRES in between) and you're interested in how the different codon makeup of the two constructs affects their expression.

[Samane]

I want to choose 2 gene of B.sub then express it to E.coli ( I should use overlab pcr to attach them together with a linker) if I want to design the primer first of all I should analysis and optimize the codon usage . coz after it I want to express them in E.coli.

[Devon Ryan]

Ah, so the overlap PCR was just for the conversion, got it. Gergana's answer would seem quite good then.

Answer 1

[Gergana Vandova]

Would you like to just analyze the codons from your B. subtilis genes or would you like to optimize them for expression in E. coli?

If you would like to just analyze them (i.e. see whether there are many rare codon that would stall expression in E. coli), I would use the Gensript rare codon analysis tool:. You have to select the organism you would like to express your gene (i.e. E. coli) and paste the open reading frame of your genes you would like to express. The output is a plot of the distribution of codon usage frequency along the length of your gene. The codon adaptation index (CAI) is correlated with the potential expression level of your gene in E. coli: the higher the CAI, the less rare codons there re and the higher the expression.

If you would like to also optimize your B. subtilis genes for E. coli expression I would recommend DNAWorks. You would just need to submit your sequence and then the software will output the optimized version of it.

[Samane]

yeah I want to optimize the B.subtilis to E.coli...thank you so much

[Gergana Vandova]

Your welcome. You might also want to upvote my answer if you found it useful. Let me know if you have issues using these tools.

Answer 2

[bioee]

To Gergana Vandova: "the higher the CAI, the less rare codons there re and the higher the expression" may be not exactly correct, the codon usage frequency is used to calculate RSCU, the RSCU is used to calculate CAI. The CAI score is calculated related to the most used synonymous codon. The optimizaed target sequence with CAI >80% is OK. If the CAI score is very high, it is NOT recommended to choose.

I used one online Rare codon analysis tool and CAI calculator with google chart plotting, it is helpful to know How-to.

For gene expression with optimization, you can choose DNAWorks, BiologicsCorp, GensScript.

PS: now most of online codon optimization tool are NOT free, I am trying to find a free one. Is there any free online codon optimization tool we can use?