Part:BBa_K1123014

DNA Coding Sequence for 1ETF Protein

This part contains the DNA sequence for the 1ETF 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 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

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 twice. The first time it was purified using a Nickel Column immediately after bugbustering the culture. A number of elution samples and a load and wash sample were taken and loaded onto an SDS gel shown below.

From the gel we can clearly see that no 1ETF protein was eluded from the Ni-column, and thus that it was either being expressed incorrectly or that it was not binding to the column correctly. From the wash sample however it is clear that there is most definitely protein being expressed, and that it is remaining within inclusion bodies. We did perform a second expression of the 1ETF protein to provide ourselves with a pellet for the MRI experiments. This time a pellet sample was loaded onto a gel alongside a supernatant sample, and again a large amount of protein was seen to be situated in the pellet.

As we could see a large band in the pellet and we still had a majority of pellet left, we bugbustered the sample and performed inclusion body extraction upon it. Unfortunately after Ni-Column purification of this solution no protein could be discovered either in the elution or wash samples, indicating that the protein had been lost along the way, most likely caused by it (possibly) being an insoluble protein.

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 a small arginine peak in the MTR_assymetric plot. But when compared to the control sample there was no detectable difference and therefore no CEST contrast.