Team:Carnegie Mellon/Met-Overview

From 2012.igem.org

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<h1>Concept</h1>
<h1>Concept</h1>
<p>
<p>
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Our synthetic reporters consist of a Spinach RNA reporter and a FAP (known as Ben) protein reporter. By placing a promoter of interest immediately upstream of Spinach, we can measure a red fluorescence signal from mRNA that is transcribed from the promoter. To measure protein expression levels, we placed a RBS at 14 base-pairs downstream of the Spinach sequence to avoid a steric clash between the Spinach RNA secondary structure and the ribosome. We also inserted our FAP after the RBS to measure protein levels over time. This way, we can measure the expression levels of both RNA and proteins in each cell over time. Our modulate construct allows the plug-and-play of different promoters, hence allowing tremendous flexibility in the characterization of any promoters. This coupled system has several advantages over a traditional system that measures protein levels through fluorescent molecules such as green fluorescent proteins (GFP), CFP, and YFP. This construct allows us to characterize more properties of any given promoter and address differences in RNA and protein expression dynamics. The plasmid map is shown here.</p>
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Our synthetic reporters consist of a Spinach RNA reporter and a FAP (known as Ben) protein reporter. By placing a promoter of interest immediately upstream of Spinach, we can measure a red fluorescence signal from mRNA that is transcribed from the promoter. To measure protein expression levels, we placed a RBS and a FAP at 14 base-pairs downstream of the Spinach sequence to avoid a steric clash between the Spinach RNA secondary structure and the ribosome. This way, we can measure the expression levels of both RNA and proteins in each cell over time. Our modular construct allows the plug-and-play of different promoters, hence allowing tremendous flexibility in the characterization of any promoters. This coupled system has several advantages over a traditional system that measures protein levels through fluorescent molecules such as green fluorescent proteins (GFP), CFP, and YFP. This construct allows us to characterize more properties of any given promoter and address differences in RNA and protein expression dynamics. The plasmid map is shown here.</p>
<p> <img src="http://igem.org/wiki/images/1/1b/CMU_plasmid_map.jpg" height="400", width="511"></img></p>
<p> <img src="http://igem.org/wiki/images/1/1b/CMU_plasmid_map.jpg" height="400", width="511"></img></p>
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<p>A simplified version of our construct is shown here.<br><img src="http://igem.org/wiki/images/a/a4/CMU_Linear_pmap.jpg" width="800", height"41"></p>
<p>A simplified version of our construct is shown here.<br><img src="http://igem.org/wiki/images/a/a4/CMU_Linear_pmap.jpg" width="800", height"41"></p>
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<h1>Design of fluorescence spectrum and measurements</h1>
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<h1>Design of fluorescence spectra and measurements</h1>
<p>
<p>
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To measure both RNA and protein levels simultaneously in a single cell, we need to design the fluorescence spectra such as the emission spectra do not overlap significantly. Therefore, we have chosen to use a FAP with Ex/Em=635/660. This FAP has a far-red emmision spectra that would be well-separated from any green fluorescence probes. For RNA measurements, we used a Spinach with Ex/Em=469/501. As shown by the following fluorescence spectra, the emmission spectra of both FAP and Spinach do not overlap.  
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To measure both RNA and protein levels simultaneously in a single cell, we need to design the fluorescence spectra such as the emission spectra do not overlap significantly. Therefore, we have chosen to use a FAP with Ex/Em=635/660. This FAP has a far-red emmision spectra that would be well-separated from any green fluorescent probes. For RNA measurements, we used a Spinach with Ex/Em=469/501. As shown by the following fluorescence spectra, the emmission spectra of both FAP and Spinach do not overlap.  
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<img src="http://2012.igem.org/wiki/images/7/7a/Spinach_Excitation-Emission.jpg", width="329"><br></p>
<img src="http://2012.igem.org/wiki/images/7/7a/Spinach_Excitation-Emission.jpg", width="329"><br></p>
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<br>
In our experiments, fluorescence intensities were measured using a Tecan SafireII at their maximum excitation and emission peaks with a 10nm bandwidth and optimal gain (100 for Spinach and 255 for the FAP).  
In our experiments, fluorescence intensities were measured using a Tecan SafireII at their maximum excitation and emission peaks with a 10nm bandwidth and optimal gain (100 for Spinach and 255 for the FAP).  
<h1>Sequences</h1>
<h1>Sequences</h1>
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The Spinach sequence we used is as follows<sup>1</sup>:
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The coding sequence of the Spinach RNA reporter is as follows<sup>1</sup>:
<br><b>GCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGAGTAATTTACGTCGACGACGCAACCGAATGAAATGGTGAAG<br>GACGGGTCCAGGTGTGGCTGCTTCGGCAGTGCAGCTTGTTGAGTAGAGTGTGAGCTCCGTAACTGGTCGCGTCG<br>ACGTCGATGGTTGCGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGGGCGCCA</b>
<br><b>GCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGAGTAATTTACGTCGACGACGCAACCGAATGAAATGGTGAAG<br>GACGGGTCCAGGTGTGGCTGCTTCGGCAGTGCAGCTTGTTGAGTAGAGTGTGAGCTCCGTAACTGGTCGCGTCG<br>ACGTCGATGGTTGCGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGGGCGCCA</b>
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The L5 sequence is as follows<sup>2</sup>:
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The coding sequence of the FAP protein reporter is as follows<sup>2</sup>:
<br><b>QAVVTQEPSVTVSPGGTVILTCGSSTGACTSGHYANWFQQKPGQAPRALIFETDKKYSWTPGRFSGSLLGAKAA<br>LTISDAQPEDEAEYYCSLSDVDGYLFGGGTQLTVLS</b>
<br><b>QAVVTQEPSVTVSPGGTVILTCGSSTGACTSGHYANWFQQKPGQAPRALIFETDKKYSWTPGRFSGSLLGAKAA<br>LTISDAQPEDEAEYYCSLSDVDGYLFGGGTQLTVLS</b>
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<br> In our experiments, we used an engineered version of the original L5 FAP that was published in Nature Biotechnology in 2008. The engineered version is easier to express in E. coli than the original. For proprietary reasons, we are giving out the modified version of the L5 construct that has already been published by Shruti et al. Bear in mind that our construct is very similar to the sequence given above and should give other labs similar results, should they choose to use this technology.
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<br> In our experiments, we used an improved version of the original L5 FAP that was published in Nature Biotechnology in 2008. The engineered version responds faster to malachite green than the L5 FAP. Due to issues of intellectual property, we are unable to deposit these reporters in the parts registry. However, we will be able to share the parts upon formal requests by other labs.
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Revision as of 21:39, 1 October 2012

Image:CMU_image6.jpeg




Concept

Our synthetic reporters consist of a Spinach RNA reporter and a FAP (known as Ben) protein reporter. By placing a promoter of interest immediately upstream of Spinach, we can measure a red fluorescence signal from mRNA that is transcribed from the promoter. To measure protein expression levels, we placed a RBS and a FAP at 14 base-pairs downstream of the Spinach sequence to avoid a steric clash between the Spinach RNA secondary structure and the ribosome. This way, we can measure the expression levels of both RNA and proteins in each cell over time. Our modular construct allows the plug-and-play of different promoters, hence allowing tremendous flexibility in the characterization of any promoters. This coupled system has several advantages over a traditional system that measures protein levels through fluorescent molecules such as green fluorescent proteins (GFP), CFP, and YFP. This construct allows us to characterize more properties of any given promoter and address differences in RNA and protein expression dynamics. The plasmid map is shown here.


A simplified version of our construct is shown here.

Design of fluorescence spectra and measurements

To measure both RNA and protein levels simultaneously in a single cell, we need to design the fluorescence spectra such as the emission spectra do not overlap significantly. Therefore, we have chosen to use a FAP with Ex/Em=635/660. This FAP has a far-red emmision spectra that would be well-separated from any green fluorescent probes. For RNA measurements, we used a Spinach with Ex/Em=469/501. As shown by the following fluorescence spectra, the emmission spectra of both FAP and Spinach do not overlap.

L5 FAP (MG) Excitation and Emission Spectra


Spinach (DFHBI) Excitation and Emission Spectra



In our experiments, fluorescence intensities were measured using a Tecan SafireII at their maximum excitation and emission peaks with a 10nm bandwidth and optimal gain (100 for Spinach and 255 for the FAP).

Sequences

The coding sequence of the Spinach RNA reporter is as follows1:
GCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGAGTAATTTACGTCGACGACGCAACCGAATGAAATGGTGAAG
GACGGGTCCAGGTGTGGCTGCTTCGGCAGTGCAGCTTGTTGAGTAGAGTGTGAGCTCCGTAACTGGTCGCGTCG
ACGTCGATGGTTGCGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGGGCGCCA

The coding sequence of the FAP protein reporter is as follows2:
QAVVTQEPSVTVSPGGTVILTCGSSTGACTSGHYANWFQQKPGQAPRALIFETDKKYSWTPGRFSGSLLGAKAA
LTISDAQPEDEAEYYCSLSDVDGYLFGGGTQLTVLS

In our experiments, we used an improved version of the original L5 FAP that was published in Nature Biotechnology in 2008. The engineered version responds faster to malachite green than the L5 FAP. Due to issues of intellectual property, we are unable to deposit these reporters in the parts registry. However, we will be able to share the parts upon formal requests by other labs.


1RNA Mimics of Green Fluorescent Protein. Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey, et al. Science 29 July 2011: 333 (6042), 642-646. [DOI:10.1126/science.1207339]
2Shruti S, Urban-Ciecko J, Fitzpatrick JA, Brenner R, Bruchez MP, et al. (2012) The Brain-Specific Beta4 Subunit Downregulates BK Channel Cell Surface Expression. PLoS ONE 7(3): e33429. doi:10.1371/journal.pone.0033429

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