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

Summary

Introduction

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.


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.


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.

Methods

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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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