Team:Carnegie Mellon/Met-Overview

From 2012.igem.org

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<h1>Concept</h1>
<h1>Concept</h1>
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The idea for the basis of promoter characterization is that our FAP (known as Ben) is a conditionally fluorescent protein. Similarly, our construct, known as Spinach is also conditionally fluorescent, but the two fluorescent constructs are not promiscuous. By placing the promoter of interest immediately upstream of Spinach, we get a fluorescence signal from the mRNA that is transcribed. To record protein fluoresence signals, we placed a RBS 14 base pairs downstream of the Spinach sequence to avoid a steric clash between the Spinach supramolecular structure and the ribosome. After the RBS, we cloned in our FAP so we can record protein levels over time as well as the RNA levels. This coupled system has several advantages over a traditional system, which only measures protein levels. This allows us to characterize more properties of any given promoter and address unpredicted behavior. 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 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>
<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>Fluorescence Readings</h1>
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<h1>Design of fluorescence spectrum and measurements</h1>
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In our experiments, fluorescence intensities were measured with 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). FAP Ex/Em=635/660 and Spinach Ex/Em=469/501.  
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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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<h3>Spinach (DFHBI) Excitation and Emission Spectra</h3>
<h3>Spinach (DFHBI) Excitation and Emission Spectra</h3>
<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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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>

Revision as of 21:30, 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 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.


A simplified version of our construct is shown here.

Design of fluorescence spectrum 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 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.

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 Spinach sequence we used is as follows1:
GCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGAGTAATTTACGTCGACGACGCAACCGAATGAAATGGTGAAG
GACGGGTCCAGGTGTGGCTGCTTCGGCAGTGCAGCTTGTTGAGTAGAGTGTGAGCTCCGTAACTGGTCGCGTCG
ACGTCGATGGTTGCGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGGGCGCCA

The L5 sequence is as follows2:
QAVVTQEPSVTVSPGGTVILTCGSSTGACTSGHYANWFQQKPGQAPRALIFETDKKYSWTPGRFSGSLLGAKAA
LTISDAQPEDEAEYYCSLSDVDGYLFGGGTQLTVLS

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.


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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