Difference between revisions of "Part:BBa K1123018"

Latest revision as of 22:56, 4 October 2013

Poly(Arginine-Glycine) Protein

This part contains the DNA sequence of a protein of our own design. We first repeated the Arginine-Glycine amino acid pair 6 times to obtain a sequence of 12 amino acids. This sequence was then repeated a total of 36 times. Between these repeats no extra amino acids were added. The idea behind this protein was to provide ourselves with a high concentration of amino acids with amide groups which we could then use to provide CEST contrast in an MRI.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 74
Illegal NotI site found at 338
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Characterization

For the iGEM 2013 Purdue team we characterized our parts according to their characterization datasheets. The data sheet for this particular biobrick can be found here.

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 50 %, 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

Lab Results

In the lab a lot of results were of course generated during the cloning of this part. For the charaterization however only the expression is of real importance. This protein was expressed once after which it entered the bugbuster protocol. A small supernatant sample was taken from this project along side a small pellet sample. These two samples were loaded onto gel so that we could analyse if any proteins had been formed:

On this gel we can see that there was no clear protein expression which was a dissapointment. No further expressions were attempted for this construct.

It should be noted however that before performing the bugbuster protocol that a small pellet sample was taken for use in the MRI experiments (where incidentally a signal was measured as you are able to read below.)

MRI 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:

Conclusion

Around 2.0 ppm there seems to be an arginine peak in the MTR_assymetric plot, but it is hard to distinguish from background noise. Also when compared to the control sample it remains unclear whether the peak is caused by Arginine or a difference in concentration between the sample and the control sample. Therefore the agent can not be clearly distinguished from the background and there is no clear CEST contrast.

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K1123018 short</partinfo>
@@ Line 14: / Line 13: @@
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K1123018 SequenceAndFeatures</partinfo>
+===Characterization===
+For the iGEM 2013 Purdue team we characterized our parts according to their characterization datasheets. The data sheet for this particular biobrick can be found [[Media:Datasheet_BBa_K1123018.pdf | here]].
+====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 '''50 %''', 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 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=====
+======Lab Results======
+In the lab a lot of results were of course generated during the cloning of this part. For the charaterization however only the expression is of real importance. This protein was expressed once after which it entered the bugbuster protocol. A small supernatant sample was taken from this project along side a small pellet sample. These two samples were loaded onto gel so that we could analyse if any proteins had been formed:
+[[File: TU-Eindhoven_Images_SP_Protein_Expression_P(RG).jpg| 150px]]
+On this gel we can see that there was no clear protein expression which was a dissapointment. No further expressions were attempted for this construct.
+It should be noted however that before performing the bugbuster protocol that a small pellet sample was taken for use in the MRI experiments (where incidentally a signal was measured as you are able to read below.)
+======MRI 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:
+[[File:TU-Eindhoven_Parts_MRI_PRG.png]]
+=====Conclusion=====
+Around 2.0 ppm there seems to be an arginine peak in the MTR_assymetric plot, but it is hard to distinguish from background noise. Also when compared to the control sample it remains unclear whether the peak is caused by Arginine or a difference in concentration between the sample and the control sample. Therefore the agent can not be clearly distinguished from the background and there is '''no''' clear CEST contrast.