Part:BBa_K1123015

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

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 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

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. The first time this protein was expressed it was bugbustered immediately after culturing had finished. This product was then purified in an attempt to obtain the protein, by making use of its His-tag and a Ni-Column. Unfortunately, upon loading a number of elution samples on a gel, along with a single load and wash sample this was not so clearly the case.

On this gel we see that there is actually no protein in any of the elution samples and that there is a small possibility of the protein being found in the wash sample. This could be caused by the proteins forming inclusion bodies after expression. The band shown in the wash sample however is not very clear or large. We decided not to continue with this protein in great detail.

A second expression of this protein was performed so that it would be possible to take a small piece of this pellet for MRI experiments. This protein was not analysed on a gel.

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 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.