Difference between revisions of "Part:BBa K4260111:Design"
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This RecA intein ESR1 biosensor consists of two inteins capable of joining and separating from two protein fragments. Considering this, the ESR1 biosensor gene coding for the hERalpha protein was introduced with its respective linker, in such a way that when an endocrine disruptor binds to the biosensor, the inteins join and separate with the rest of the protein, removing the linker, the biosensor and the two inteins. The two endogenous fragments encode the chromoprotein AmilCP [<html><a href='https://parts.igem.org/Part:BBa_K592009'>BBa_K592009</a></html>]. | This RecA intein ESR1 biosensor consists of two inteins capable of joining and separating from two protein fragments. Considering this, the ESR1 biosensor gene coding for the hERalpha protein was introduced with its respective linker, in such a way that when an endocrine disruptor binds to the biosensor, the inteins join and separate with the rest of the protein, removing the linker, the biosensor and the two inteins. The two endogenous fragments encode the chromoprotein AmilCP [<html><a href='https://parts.igem.org/Part:BBa_K592009'>BBa_K592009</a></html>]. | ||
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| + | RecA intein was build from the first 110 and last 58 aminoacids from the wildtype RecA full-lenght intein [1]. Moreover two aminoacid mutationes (Val67Leu and Asp111Glu) to make the RecA intein a stable protein [2]. | ||
| + | The functionality of the Barcelona 2020 team's inteins from the biobrick [<html><a href='https://parts.igem.org/Part:BBa_K3484000'>BBa_K348400</a></html>]. was verified to check the functibility of the HERα enzyme to be used in the biosensor, since it is employed as a receptor enzyme for EDCs. | ||
| + | Moreover, the wildtype RecA intein sequence was obtained from Novel Structure of the recA Locus of Mycobacterium tuberculosis (Davis, E., Sedgwick, S., & Colston, J. 1991); only the first 110 and last 58 coding amino acids were used to build the RecA intein mediated biosensor. A change of an amino acid and elimination of a proline at the end of the intein was performed, because according to studies by David W. Wood (1999) et al. and Izabela Gierach (2012) et al, this modification allowed storing the precursor for several days at 4°C and pH 8.5, this was also evaluated at other conditions (such as pH 5 and 4, and temperature of 34°C) and there was not found significant scission or loss of activity occurred. We are interested in this because of the nature of the biosensor as it is in contact with residual water used to irrigate agricultural crops. Likewise, the biobrick from Barcelona 2020 team had a reporter gene that was replaced by the reporter enzyme BBa_K592009 from the Uppsala-Sweden 2011 team. | ||
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[[File:RecAintESR1_CDS_diagram.png|350px|]] | [[File:RecAintESR1_CDS_diagram.png|350px|]] | ||
Revision as of 21:19, 30 September 2022
Design
This RecA intein ESR1 biosensor consists of two inteins capable of joining and separating from two protein fragments. Considering this, the ESR1 biosensor gene coding for the hERalpha protein was introduced with its respective linker, in such a way that when an endocrine disruptor binds to the biosensor, the inteins join and separate with the rest of the protein, removing the linker, the biosensor and the two inteins. The two endogenous fragments encode the chromoprotein AmilCP [BBa_K592009].
RecA intein was build from the first 110 and last 58 aminoacids from the wildtype RecA full-lenght intein [1]. Moreover two aminoacid mutationes (Val67Leu and Asp111Glu) to make the RecA intein a stable protein [2].
The functionality of the Barcelona 2020 team's inteins from the biobrick [BBa_K348400]. was verified to check the functibility of the HERα enzyme to be used in the biosensor, since it is employed as a receptor enzyme for EDCs.
Moreover, the wildtype RecA intein sequence was obtained from Novel Structure of the recA Locus of Mycobacterium tuberculosis (Davis, E., Sedgwick, S., & Colston, J. 1991); only the first 110 and last 58 coding amino acids were used to build the RecA intein mediated biosensor. A change of an amino acid and elimination of a proline at the end of the intein was performed, because according to studies by David W. Wood (1999) et al. and Izabela Gierach (2012) et al, this modification allowed storing the precursor for several days at 4°C and pH 8.5, this was also evaluated at other conditions (such as pH 5 and 4, and temperature of 34°C) and there was not found significant scission or loss of activity occurred. We are interested in this because of the nature of the biosensor as it is in contact with residual water used to irrigate agricultural crops. Likewise, the biobrick from Barcelona 2020 team had a reporter gene that was replaced by the reporter enzyme BBa_K592009 from the Uppsala-Sweden 2011 team.
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