Difference between revisions of "Part:BBa K1951008:Design"

Revision as of 22:08, 15 October 2016

High level flagellin expression: This biobrick is composed of two parts:

• BBa_K880005 A strong promoter, strong RBS combination specifically conceived and tested for high expression levels of proteins in E.coli (this part is itself composed of sub-parts BBa_J23100 and BBa_B0034
• BBa_K1951005 the FliC coding sequence designed by our team for high level expression in E.coli with sequence optimization.

Optimization of Bba_K1951008

Sequence optimization

This part is optimized for expression in E.coli by codon optimization and synthesis of a synthetic gene. Codon optimization was done using the tool provided by IDT ^[1]. Codon optimization has been shown to improve protein expression by increasing the translational efficiency of gene of interest, and consists of choosing for each amino acid the codon that works best in the target species Claire a ref or 2. We optimized the sequence for expression in E.coli.

In addition to changing the codons from the E.coli sequence we also ensured that all forbidden restriction sites were absent to ensure compatibility with the biobrick standard. We also designed the insertion of a Bbs1 site at amino acid 130 to allow insertion of additional sequences in the loop at this position in the sequence. Unfortunately we did not get round to using this feature.

Promoter and RBS selection

As the purpose of this part is high level protein expression we selected the part BBa_K880005 to drive expression of our coding sequence. We chose this part as:

• it has already been successfully used in the past,
• it was available in the distribution,
• it combines a strong promotor and a RBS (so avoiding the separate incorporation of 2 parts).

Source

We constructed this part from 2 others :

• BBa_K1951005
• BBa_K880005

References

1. ↑ https://eu.idtdna.com/CodonOpt