Difference between revisions of "Team:Glasgow/Project/Overview/UVA"
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Due to possible toxicity of the UirS gene, our proposed construct contains UirR and PlsiR with an appropriate coding gene in the high copy number pSB1C3, while UirS, alongside with the phycocyanobillin synthesis operon, was put in the low copy number plasmid pSB3K3. We have decided not to put a terminator between UirS and K322122 because the promoter of K322122 is stronger than that of UirS. | Due to possible toxicity of the UirS gene, our proposed construct contains UirR and PlsiR with an appropriate coding gene in the high copy number pSB1C3, while UirS, alongside with the phycocyanobillin synthesis operon, was put in the low copy number plasmid pSB3K3. We have decided not to put a terminator between UirS and K322122 because the promoter of K322122 is stronger than that of UirS. | ||
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Revision as of 21:56, 12 September 2015
Summary
Aims:
- To characterise the three components of a UV-A sensor system: UirS, UirR, PlsiR.
- To investigate E. coli survival rates in UV-A and sunlight.
- Essential: • K1725400 (PlsiR) • K1725410 (UirS) • K172541#(UirS with RBS) • K1725420 (UirR) • K1725421 (UirR with RBS)
- Others: • K1725401 (PlsiR.I13500) • K1725402 (PlsiR.E5501) • K1725422 (J23101.B0032.UirR) • K1725423 (J23110.B0032.UirR) • K1725424 (J23114.B0032.UirR) • K1725425 (J23116.B0032.UirR) • K1725426 (J23101.B0032.UirR.B0015) • K1725427 (J23110.B0032.UirR.B0015) • K1725428 (J23114.B0032.UirR.B0015) • K1725429 (J23116.B0032.UirR.B0015) • K1725430 (J23101.B0032.UirR.B0015.PlsiR.I13500) • K1725431 (J23110.B0032.UirR.B0015.PlsiR.I13500) • K1725432 (J23114.B0032.UirR.B0015.PlsiR.I13500) • K1725433 (J23116.B0032.UirR.B0015.PlsiR.I13500)
Overview
In order to prevent cells permanently shifting their metabolism towards one that favours light production, we decided to repress the bioluminescence genes (our optimised lux operon) in the presence of sunlight. The chosen system recognizes the presence of unidirectional UV-A light and comes from the cyanobacterium Synechocystis sp. PCC6803. The system has not been previously characterized before in iGEM. Three components are required to produce a response to UV-A: UirS (UV intensity response Sensor), UirR (UV intensity response Regulator), and PlsiR (promoter of the light and stress integrating response Regulator). For the system to be fully online K322122 is required. This BioBrick is responsible for the synthesis of phycocyanobillin, a chromophore normally found in cyanobacteria that is necessary for the functioning of nearly all light-sensing proteins.
We obtained UirS and UirR from genomic Synechocystis DNA via PCR. Our primers included the BioBrick prefix and suffix as well as a ribosome-binding site (B0032). Due to non-BioBrick compatible restriction sites in the UirS gene PCR mutagenesis was carried out with the use of the TOPO TA plasmid vector. The UirR gene contained no such sites and was therefore inserted directly into the pSB1C3 plasmid. PlsiR also lacked such restriction sites and was therefore inserted into pSB1C3.
Due to possible toxicity of the UirS gene, our proposed construct contains UirR and PlsiR with an appropriate coding gene in the high copy number pSB1C3, while UirS, alongside with the phycocyanobillin synthesis operon, was put in the low copy number plasmid pSB3K3. We have decided not to put a terminator between UirS and K322122 because the promoter of K322122 is stronger than that of UirS.
Methods
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque
Results
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Nam fermentum, nulla luctus pharetra vulputate, felis tellus mollis orci, sed rhoncus sapien nunc eget odio. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean euismod bibendum laoreet. Proin gravida dolor sit amet lacus accumsan et viverra justo commodo. Proin sodales pulvinar tempor. Cum sociis natoque
Conclusion
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