Feb 4, 2010

ATG:Newsletter 100204

Assembly Cloning for Single and Multiple Co-Expression by use of Modular Expression Cassettes - the Perfect Functional Environment for Synthetic Genes

Transcription and Translation Initiation are the rate limiting steps in expression of genes.

Translational elongation can only be as processive as the initiation frequency provides sufficient ribosomes for an efficient elongation process. Other factors influencing the fidelity of the expression process as a whole are the stability of the messenger RNA and the formation of poly-ribosomal structures for the availability of correct folded active protein.

Coding sequences of genes (CDS) are embedded in a formal-operational genetic frame consisting of a non-transcribed promotor region were transcription is initiated. The transcribed part of the operational frame is divided in a non-translated 5'-trailer sequence followed by the coding sequence CDS and a 3'-non-translated trailers sequence.

It was shown in a variety of publications that the 5'-non-translated trailer sequence flanking the ribosomal binding site (Shine Dalgarno, Koszak) concomitant with the 5'-translated sequences of the message on the mRNA level are contributing to a high extent to the expression rate of genes (Kudla et al. (2009), Salis et al. (2009)).

The reason for this are highly dynamic formations of secondary structures having the potential for preventing the ribosomal anti-RBS-rRNA sequences from the intermolecular interaction in order to initiate the translational process efficiently. Expression optimization without the extensive reduction of rate limiting intra-molecular 5'-non-translated mRNA- secondary structures makes no sense and is not feasible.

The 5'-non-translated trailer is a constant and predefined expression vectors (e.g. the pET-series) show virtually no predictable behavior in respect to expression. The fixed 5'-trailers of expression vectors in combination with the constraints in the freedom of design of the 5'-translated sequence of the CDS is a random combination which sometimes promotes translation initiation sometimes it is neutral but sometimes it does not work.

This findings support strongly the ACEMBL - expression cassette model for designing sequences for formal-operational smooth expression conditions.

Calculating out secondary structures in both: the 5'- non-translated-trailer and the 5'- coding sequence of genes (CDS) is more predictable in respect to reliable efficient translation initiation the rate limiting step in the whole process.

The ATG: evoMAG - in silico evolution machine is capable for calculating out any secondary structures from DNA/ RNA sequences - especially exactly those which are relevant for translational initiation. But in addition it is capable for simultaneously controlling the use of frequent codons and those having high of probability incorporation rates (Fluitt A. et al. (2007)) and taking in account the codon distribution reflecting indirectly the amino-acyl-tRNA-pool sizes.

Please use our gene check service for free by just submitting the sequence of your interest and get a feedback from ATG with an predictive assessment of your sequence.

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May 7, 2013
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PepID bio-based Peptide Libraries:



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