Part:BBa_K1123003

1PJN Protein Production Construct

This composite part has been built up from a number of separate BioBricks, the most prominent of which the FNR promotor and the 1PJN Protein sequence. The FNR promotor sequence enables us to bring the 1PJN protein to expression anaerobically. Almost all E.coli strains posses a form of this promotor to induce metabolic processes once the bacteria enters anaerobic regions. The 1PJN protein is a protein which we are expressing with the sole purpose of using it as a CEST contrast agent.

TU-Eindhoven Sequencing Results

This construct has been submitted to the iGEM registry and has been privately sequenced by the iGEM 2013 TU-Eindhoven. The sequencing results were positive indicating that the sequence and part submitted to the registry are correct. Sequencing was performed by BaseClear Netherlands

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 200
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 39
  Illegal XhoI site found at 596
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

This part is a composite part which is comprised of a number of other important parts namely BBa_K1123000 and BBa_K1123015.

This part has, in itself not been tested by us here at the TU-Eindhoven during the 2013 iGEM competition. However, as the other, important parts which make up this part have been tested, shown to work, and characterized we would like to refer you to these parts for a complete overview of the characterization of this part.

We do not think there will be a change in the functionality of any of the separate parts when combining them. A similar composite part BBa_K1123005 was combined and tested and showed no change in functionality other than a decrease in expression.

Further characterization of the simple parts

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.