Difference between revisions of "Part:BBa K1123013:Experience"

Revision as of 12:38, 4 October 2013

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Applications of BBa_K1123013

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 Arginine percentage of 48 %, 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].

User Reviews

UNIQa6b70517a9b216d5-partinfo-00000000-QINU UNIQa6b70517a9b216d5-partinfo-00000001-QINU

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 =====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.
+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.
 =====Results=====