In a previous project, the apparent lack of specificity of the promoter BioBrick we looked to improve upon proved to be a minor difficulty that we felt had not been addressed previously in the Registry. Thus we decided to tackle the issue by devising a way of quantitatively measuring the output of NO with the non-specific promoter we were using through a novel gene regulation system.
In a previous project, the apparent lack of specificity of the promoter BioBrick we looked to improve upon proved to be a minor difficulty that we felt had not been addressed previously in the Registry. Thus we decided to tackle the issue by devising a way of quantitatively measuring the output of NO with the non-specific promoter we were using through a novel gene regulation system.
Assembling the various gene constructs was not without its challenges. The construction of complimentary ‘zips’ within the sequence that surrounded and, in the case of the contracurrent comparator circuit, included the ribosome binding site it was often the case that the DNA sequence would form unwanted secondary structures. When designing the DNA we took care to avoid these structures obstructing sequences required for translation of the mRNA and, at the same time, only using codons that we know are not rare in E. coli and that code for an amino acid that is unlikely to change the function of the protein produced. This is required since the zip sequences extend past the translational start codon, thus our construct will add a small N-terminal tag to the reporter protein.
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Due to the stop codon present in the scars of Assembly Standard 10 BioBricks, we decided that our constructs would have to be an Assembly Standard 23 BioBrick. Although the use of Bioscaffolds produced by previous iGEM teams was considered, time constraints meant that changing the Assembly Standard we put our BioBricks in would be the most convenient solution.
==Experiments==
==Experiments==
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. Ultimately ligate with NO-sensing promoters and effecter enzymes to control NO levels/reporters to detect NO levels quantitatively
. Ultimately ligate with NO-sensing promoters and effecter enzymes to control NO levels/reporters to detect NO levels quantitatively
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==Theoretical characterisation==
==Future Applications==
==Future Applications==
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DONT READ THIS!!
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Assembling the various gene constructs was not without its challenges. The construction of complimentary ‘zips’ within the sequence that surrounded and, in the case of the contracurrent comparator circuit, included the ribosome binding site it was often the case that the DNA sequence would form unwanted secondary structures. When designing the DNA we took care to avoid these structures obstructing sequences required for translation of the mRNA and, at the same time, only using codons that we know are not rare in E. coli and that code for an amino acid that is unlikely to change the function of the protein produced. This is required since the zip sequences extend past the translational start codon, thus our construct will add a small N-terminal tag to the reporter protein.
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Due to the stop codon present in the scars of Assembly Standard 10 BioBricks, we decided that our constructs would have to be an Assembly Standard 23 BioBrick. Although the use of Bioscaffolds produced by previous iGEM teams was considered, time constraints meant that changing the Assembly Standard we put our BioBricks in would be the most convenient solution.
Biological systems function on a great variety of different integrative mechanisms which include post-transcriptional attenuation. We believe that Synthetic Biology is at its most effective when these natural mechanisms are taken and applied in novel situations. This ethos we have sought to emulate by creating our own mechanism of post-transcriptional attentuation; the Comparator Circuit.
In a previous project, the apparent lack of specificity of the promoter BioBrick we looked to improve upon proved to be a minor difficulty that we felt had not been addressed previously in the Registry. Thus we decided to tackle the issue by devising a way of quantitatively measuring the output of NO with the non-specific promoter we were using through a novel gene regulation system.
Planning
Assembling the various gene constructs was not without its challenges. The construction of complimentary ‘zips’ within the sequence that surrounded and, in the case of the contracurrent comparator circuit, included the ribosome binding site it was often the case that the DNA sequence would form unwanted secondary structures. When designing the DNA we took care to avoid these structures obstructing sequences required for translation of the mRNA and, at the same time, only using codons that we know are not rare in E. coli and that code for an amino acid that is unlikely to change the function of the protein produced. This is required since the zip sequences extend past the translational start codon, thus our construct will add a small N-terminal tag to the reporter protein.
Due to the stop codon present in the scars of Assembly Standard 10 BioBricks, we decided that our constructs would have to be an Assembly Standard 23 BioBrick. Although the use of Bioscaffolds produced by previous iGEM teams was considered, time constraints meant that changing the Assembly Standard we put our BioBricks in would be the most convenient solution.
Experiments
. Designed DNA constructs for the subtractive system and had them synthesised
. Made into biobricks (link to registry page)
Future Experiments
. Ligating with different promoters and reporters to test
. Ultimately ligate with NO-sensing promoters and effecter enzymes to control NO levels/reporters to detect NO levels quantitatively
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