Difference between revisions of "Part:BBa K1123020"

Latest revision as of 22:58, 4 October 2013

Poly(Threonine-Lysine) Protein

This part contains the DNA sequence of a protein of our own design. We first repeated the Threonine-Lysine 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. The DNA sequence can be placed behind any compatible promoter and the protein will be expressed.

Sequence and Features

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 Lysine 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 characterization 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 disappointment. 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 3.7 ppm is no clear Lysine peak distinguishable. Although in the difference plots there is a high signal at the specific chemical shift, it remains unclear whether this peack is caused by the abundance of Lysines or just background noise. So there is no clear CEST contrast observed yet.