Part:BBa_K1123016

DNA Coding Sequence for 1G70 Protein

This part contains the DNA sequence for the 1G70 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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 64
  Illegal NgoMIV site found at 148
  Illegal NgoMIV site found at 232
  Illegal NgoMIV site found at 316
  
• 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 46 %, 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 1G70 protein was expressed once. After protein expression had taken place the sample was spun down and a small pellet portion was loaded onto an SDS gel alongside a portion of the supernatant:

Here we can clearly see a large amount of protein in the pellet which indicates that the protein is being held in inclusion bodies. As only a small portion of the pellet had been used for the above gel analysis the rest was treated making use of an inclusion body extraction kit. After being treated as such it was loaded onto a Ni-Column for purification. During the Ni-Column purification elution samples were captured along with a wash and load sample. These samples were subsequently loaded onto a second SDS gel and run giving the following results:

On the above gel it is obvious that the 1G70 protein has been formed and that inclusion body extraction has been successful.

A second expression was performed so that a small pellet portion could be used for the MRI experiments. These pellets were not bugbustered, only spundown after expression.

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.