Difference between revisions of "Part:BBa K1123015"

 
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<partinfo>BBa_K1123015 short</partinfo>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1123015 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1123015 SequenceAndFeatures</partinfo>
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===Characterization===
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====Characterization by the TU Eindhoven 2013 iGEM Team====
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This part was designed to generate CEST MRI contrast. The basic principle behind this technique is based on compounds that contain pools of exchangeable protons that can be selectively saturated using radiofrequency irradiation. Upon proton exchange with bulk water, these compounds can be indirectly visualized by measuring the bulk water using an MRI machine. The amino acids Lysine, Arginine, Threonine and Serine contain those exchangeable protons and polypeptides containing those amino acids in abundance are therefore potential contrast agents (see also [http://2013.igem.org/Team:TU-Eindhoven/Background CEST 101]).
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The protein of this part has a ''Lysine'' percentage of '''38 %''', which is high compared to other (native) proteins. Therefore, it was expected that this protein would be detectable using CEST MRI.
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=====Methods=====
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The proteins were (aerobically) overexpressed in BL21 using a pET28a vector with a T7 promotor. The bacteria were spun down and fixed in PFA. The entire pellet (bacteria containing our proteins) was then measured in a 7 T Bruker MRI machine. First, the correct water frequency was determined, the machine was shimmed, i.e. a homogeneous magnetic field was created. The first measurement was a T<sub>2</sub> weighed image for general orientation. Subsequently local shimming was performed on each of the separate pellets. For the final measurements, the saturation pulse was set to vary from ca. -4ppm to ca. +4ppm (relative to water), the measurements were averaged over 8 separate scans. Also a S<sub>0</sub> (without saturation pulse) image was taken.
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=====Results=====
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This data was processed resulting in a asymmetry plot (MTR_assymetric) and a contrast plot visualizing the difference of the sample with the control sample. This was done by both subtracting (absolute difference) and dividing (relative difference). For a complete overview of the processing steps see the TU-Eindhoven 2013 [http://2013.igem.org/Team:TU-Eindhoven/MRIProcessing MRI Data Processing page]. The results are shown below:
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[[File:TU-Eindhoven_Parts_MRI_1PJN.png]]
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=====Conclusion=====
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Around 3.7 ppm there is a clear lysine peak in the MTR_assymetric plot. Especially when compared to the control sample the lysine peak specifically stands out and therefore '''this part does generate (lysine based) CEST contrast'''.
  
  

Revision as of 14:22, 4 October 2013

DNA Coding Sequence for 1PJN Protein

This part contains the DNA sequence for the 1PJN protein. It can be placed behind any promoter of your choice and expressed with ease. Within the scope of our project this protein was expressed due to its high Arginine and Lysine concentration. This could then be used to provide CEST contrast within an MRI.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

Characterization by the TU Eindhoven 2013 iGEM Team

This part was designed to generate CEST MRI contrast. The basic principle behind this technique is based on compounds that contain pools of exchangeable protons that can be selectively saturated using radiofrequency irradiation. Upon proton exchange with bulk water, these compounds can be indirectly visualized by measuring the bulk water using an MRI machine. The amino acids Lysine, Arginine, Threonine and Serine contain those exchangeable protons and polypeptides containing those amino acids in abundance are therefore potential contrast agents (see also [http://2013.igem.org/Team:TU-Eindhoven/Background CEST 101]).

The protein of this part has a Lysine percentage of 38 %, which is high compared to other (native) proteins. Therefore, it was expected that this protein would be detectable using CEST MRI.

Methods

The proteins were (aerobically) overexpressed in BL21 using a pET28a vector with a T7 promotor. The bacteria were spun down and fixed in PFA. The entire pellet (bacteria containing our proteins) was then measured in a 7 T Bruker MRI machine. First, the correct water frequency was determined, the machine was shimmed, i.e. a homogeneous magnetic field was created. The first measurement was a T2 weighed image for general orientation. Subsequently local shimming was performed on each of the separate pellets. For the final measurements, the saturation pulse was set to vary from ca. -4ppm to ca. +4ppm (relative to water), the measurements were averaged over 8 separate scans. Also a S0 (without saturation pulse) image was taken.

Results

This data was processed resulting in a asymmetry plot (MTR_assymetric) and a contrast plot visualizing the difference of the sample with the control sample. This was done by both subtracting (absolute difference) and dividing (relative difference). For a complete overview of the processing steps see the TU-Eindhoven 2013 [http://2013.igem.org/Team:TU-Eindhoven/MRIProcessing MRI Data Processing page]. The results are shown below: TU-Eindhoven Parts MRI 1PJN.png

Conclusion

Around 3.7 ppm there is a clear lysine peak in the MTR_assymetric plot. Especially when compared to the control sample the lysine peak specifically stands out and therefore this part does generate (lysine based) CEST contrast.