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Team:Warsaw/ExpressionAdaptors/Solution
From 2011.igem.org
Our Solution
To make truly standardize strength of RBS parts we propose a brand new part in synthetic biology - we call expression adapters. They are short sequences consisting of RBS, 5 bp spacer and a short (10 amino acid long) beginning of a protein. An expression adapter look like this:
Why are they constructed this way:
Spacer and short protein were created by a genetic algorithm that we designed specifically for this task. It generates random 5 bp spacers and 10 amino acid long proteins. Proteins consist of only 11 amino acids, those that does not direct to the N- degradation pathway [1]. Genetic algorithm was used because there is too many combinations of random spacers and 10 aa proteins to test them all. First generation of spacers and proteins is submitted to several rounds of random mutations and recombination. The outcome sequences from all the rounds of genetic alterations were then gathered in one list.
After removal of the duplicates sequences were scored using RBS calculator – an algorithm developed by Christopher A. Voigt [2]. The calculator considers parameters like fold of the protein, complementarity with the ribosome and... Each adapter sequence is modeled with several fluorescent proteins, namely GFP, SF-GFP, YFP, mORANGE and RFP. Those with the best score are then processed with another software tool that chooses adapters with the best (highest) expression levels and lowest deviations between different proteins.
From the final forty sequences six adapters providing six different levels of expression were chosen manually and synthesized in Laboratory of DNA Sequencing and Oligonucleotide Synthesis in the Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland). The final step was to test them in the wet lab.
Why are they constructed this way:
- RBS-spacer-and-the-beginning-of-a-protein determin the fold of the mRNA around the RBS. This way the fold, and subsequently the strength of the RBS is independent of the protein used.
- Spacer is a piece of DNA that is between RBS (e.g. B0034) and ORF (open reading frame). It keeps the optimal distance between those two parts - needed for efficient protein expression
- Promoter is not a part of expression adapter because it is a DNA sequence and is not translated to mRNA
Spacer and short protein were created by a genetic algorithm that we designed specifically for this task. It generates random 5 bp spacers and 10 amino acid long proteins. Proteins consist of only 11 amino acids, those that does not direct to the N- degradation pathway [1]. Genetic algorithm was used because there is too many combinations of random spacers and 10 aa proteins to test them all. First generation of spacers and proteins is submitted to several rounds of random mutations and recombination. The outcome sequences from all the rounds of genetic alterations were then gathered in one list.
After removal of the duplicates sequences were scored using RBS calculator – an algorithm developed by Christopher A. Voigt [2]. The calculator considers parameters like fold of the protein, complementarity with the ribosome and... Each adapter sequence is modeled with several fluorescent proteins, namely GFP, SF-GFP, YFP, mORANGE and RFP. Those with the best score are then processed with another software tool that chooses adapters with the best (highest) expression levels and lowest deviations between different proteins.
From the final forty sequences six adapters providing six different levels of expression were chosen manually and synthesized in Laboratory of DNA Sequencing and Oligonucleotide Synthesis in the Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland). The final step was to test them in the wet lab.
