Difference between revisions of "Team:Glasgow/Project/Overview/UVA"

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             <p class="mainText">We needed a repressor in our genetic circuit to act as an inverter – ie our UVA sensor turns on transcription, but we needed to to turn off transcription when UVA was present. There are several repressor protein/repressible promoter pairs in the iGEM registry such as TetR or LacI, however, Stanton et al (2014) have recently identified 16 prokaryotic TetR-like repressors by genomic mining and designed synthetic repressible promoters. To understand how they were able to design synthetic repressible promoters, it is important to understand how promoters and repressors work. We are all familiar with the central dogma of molecular biology: DNA to RNA to protein. Transcription is the process where RNA polymerase binds to DNA to make mRNA – a promoter tells RNA polymerase where to bind to the DNA, so a promoter is found upstream of a gene.  
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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.
 
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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.
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Promoters have -10 and -35 sites that RNA polymerase recognises – and transcription starts at “0” ie 10 bases after the -10 site and 35 bases after the -35 site, hence the names of the sites. If one or both of these sites are bound by another protein, RNA polymerase cannot recognise the promoter, and transcription does not take place. Repressors  are proteins that bind to DNA at a specific sequence; this is called the operator sequence. Stanton et al (2014) overlapped the operator sequence for each repressor over the -10 and/or -35 sites of BBa_J23119, a constitutive promoter. This means that when the repressor binds to it’s operator sequence, RNA polymerase cannot recognise the promoter, and transcription cannot start. This is how the synthetic repressible promoters work. For a repressor to be useful in a genetic circuit, is must be specific so as not to interfere with another part of the circuit or have unwanted interactions within the cell. Repressors that do this are called orthogonal – ie repressor A binds to promoter A; repressor B binds to promoter B; but repressor A cannot bind to promoter B, and vice versa. 
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We decided to make BioBricks of 2 of the 16 repressors and characterise them for the iGEM registry. The first repressor we decided to make into a BioBrick was the PhlF repressor from Pseudomonas protegens Pf-5. In P. protegens Phlf represses the phlACBD operon which synthesises the 2,4-diacetylphloroglucinol (PHL); which in turn interacts with PhlF to prevent it binding to operator sequence. The second repressor was the SrpR repressor from Pseudomonas putida S12. In P. putida SrpR represses something. We aimed to characterise both PhlF and SrpR and their respective repressible promoters.<div style="visibility:hidden; height:0;width:0;" class="scrollSurvivability"></div> </p>
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         <h2>Methods</h2>
 
         <h2>Methods</h2>

Revision as of 21:52, 12 September 2015

Home > Project >UVA

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.

Results:
Parts:
  • 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.

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

Read More!

Protocols

Bioluminesence

Steve

Repressors

Location

Bower Building, Wilkins Teaching Laboratory
University of Glasgow
University Avenue
G12 8QQ

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