Difference between revisions of "Team:Glasgow/Project/Overview/Repressors"
| Line 214: | Line 214: | ||
<h2>Introduction</h2> | <h2>Introduction</h2> | ||
| − | <p class="mainText"> | + | <p class="mainText">Our genetic circuit needed an inverter, as our UVA sensor turns on transcription, but our circuit needed to turn off transcription when UVA was present. There are several repressor protein/repressible promoter pairs in the iGEM registry suitable for with function such as TetR or LacI, however, it was decided to characterise and submit two new repressors to the registry. Stanton et al., (2014) have recently identified sixteen prokaryotic TetR-like repressors by genomic mining and designed synthetic repressible promoters, as shown in Figure 1A. To understand how they were able to design synthetic repressible promoters, it is important to understand how promoters and repressors work. |
</br> | </br> | ||
</br> | </br> | ||
</br> | </br> | ||
| − | Promoters have -10 and -35 sites that RNA polymerase recognises | + | 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, as shown in Figure1B, transcription starts at +1. If one or both of these sites are bound by another protein, RNA polymerase cannot recognise the promoter, and transcription does not take place. Transcriptional 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 strong, constitutive Anderson family promoter, meaning when the repressor binds to its operator sequence RNA polymerase cannot recognise the promoter and transcription cannot start, as shown in Figure 1B. This is how the synthetic repressible promoters work. |
</br> | </br> | ||
</br> | </br> | ||
</br> | </br> | ||
| − | + | 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. Repressor A binds to promoter A; repressor B binds to promoter B; but repressor A cannot bind to promoter B, and vice versa. The sixteen prokaryotic TetR-like repressors Stanton et al., (2014) identified are orthogonal, as shown in Figure 2. In particular, TetR, PhlF, and SrpR do not show significant repression of the other’s repressible promoters. | |
| + | </br> | ||
| + | </br> | ||
| + | </br> | ||
| + | It was decided to make BioBricks of two of the sixteen repressors and characterise them for the iGEM registry. The first repressor we decided to submit as a BioBrick was the PhlF repressor from <i>Pseudomonas protegens Pf-5</i>. In <i>P. protegens</i> PhlF is involved in regulation of the <i>phlACBD</i> operon which synthesises an antifungal metabolite 2,4-diacetylphloroglucinol (PHL). (Sheehan et al., 2000, Abbas et al., 2002) The second repressor was the SrpR repressor from <i>Pseudomonas putida S12</i>. In <i>P. putida</i> SrpR is involved in regulation of the <i>srpABC</i> operon which is involved in organic solvent tolerance. (Wery et al., 2001, Sun et al., 2011) The aim was to submit and characterise both <i>phlF</i> and <i>srpR</i> and their respective repressible promoters.<div style="visibility:hidden; height:0;width:0;" class="scrollSurvivability"></div> </p> | ||
<h2>Methods</h2> | <h2>Methods</h2> | ||
Revision as of 13:56, 15 September 2015
Summary
Introduction
Our genetic circuit needed an inverter, as our UVA sensor turns on transcription, but our circuit needed to turn off transcription when UVA was present. There are several repressor protein/repressible promoter pairs in the iGEM registry suitable for with function such as TetR or LacI, however, it was decided to characterise and submit two new repressors to the registry. Stanton et al., (2014) have recently identified sixteen prokaryotic TetR-like repressors by genomic mining and designed synthetic repressible promoters, as shown in Figure 1A. To understand how they were able to design synthetic repressible promoters, it is important to understand how promoters and repressors work. 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, as shown in Figure1B, transcription starts at +1. If one or both of these sites are bound by another protein, RNA polymerase cannot recognise the promoter, and transcription does not take place. Transcriptional 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 strong, constitutive Anderson family promoter, meaning when the repressor binds to its operator sequence RNA polymerase cannot recognise the promoter and transcription cannot start, as shown in Figure 1B. 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. Repressor A binds to promoter A; repressor B binds to promoter B; but repressor A cannot bind to promoter B, and vice versa. The sixteen prokaryotic TetR-like repressors Stanton et al., (2014) identified are orthogonal, as shown in Figure 2. In particular, TetR, PhlF, and SrpR do not show significant repression of the other’s repressible promoters. It was decided to make BioBricks of two of the sixteen repressors and characterise them for the iGEM registry. The first repressor we decided to submit as a BioBrick was the PhlF repressor from Pseudomonas protegens Pf-5. In P. protegens PhlF is involved in regulation of the phlACBD operon which synthesises an antifungal metabolite 2,4-diacetylphloroglucinol (PHL). (Sheehan et al., 2000, Abbas et al., 2002) The second repressor was the SrpR repressor from Pseudomonas putida S12. In P. putida SrpR is involved in regulation of the srpABC operon which is involved in organic solvent tolerance. (Wery et al., 2001, Sun et al., 2011) The aim was to submit and characterise both phlF and srpR and their respective repressible promoters.
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!
