Part:BBa_K3139014

J23100-fusion protein-B0015-J23106-TEVp-TEVp site-HasA-B0010 anti-plasmodium system

Usage and Biology

In the anti-plasmodium system of NAU_2019, the amount of both TEV protease BBa_K3139012and anti-plasmodium fusion protein BBa_K3139013should be kept at a certain proportion to avoid a waste of cell resources. If the TEV protease is excessive, it shall be wasted though the fusion proteins are cleaved completely. If the fusion protein is excessive, it cannot be cleaved completely with insufficient TEV protease. By testing different promoter combinations of TEVp and fusion protein, we tried to find out the most economical combination which has the least waste and most complete cleavage with the help of mathematic model. To measure, we planned to replace the promoter of TEVp, and in this part we chose promoter J23106, and acquired the best effect among all 3 promoters.

Characterization of BBa_K3139014

Background

Composite part BBa_K3139014, J23100-fusion protein-B0015-J23106-TEVp-TEVp site-HasA-B0010, anti-Plasmodium system. We choose this part as the best composite part, because its function was the most superior among all the parts of the same type (Fig.3 Lane 3). It also provided significant data for our mathematic model which simulated different promoter combinations (shown in Figure.1) of anti-Plasmodium system and predicted the possibly most superior combination. the amount of both TEV protease and anti-Plasmodium fusion protein should be kept at a certain proportion to avoid a waste of cell resources. If the TEV protease is excessive, it shall be wasted though the fusion proteins are cleaved completely. If the fusion protein is excessive, it cannot be cleaved completely with insufficient TEV protease.

Fig.1 Recombinant plasmid used

Fig.2 Mechanism of anti-Plasmodium system

1.Anti-Plasmodium fusion protein.

2.TEV protease with hasA signal peptide, unfolded and nonfunctioning.

3.1 and 2 are secreted by has secretion complex.

4.Anti-Plasmodium fusion protein is cleaved by TEV protease and turns functioning.

5.HasA signal peptide is cutted off, the TEV protease is completely functioning. The protease is mostly folded and functioning when it is secreted out of the cell.

Characterization

We constructed a recombined plasmid vector pEXC-2 (Fig.1), transformed the plasmid into Serratia JCM11315, and cultured it in LB medium for 60h to obtain supernatant after ultrasonication. Then we purified our target His tagged protein by using Ni-NTA magnetic beads.

Then we made mass spectrometry analysis.

Results

The WB result shows several bands (Figure.3). We defined the smallest monomer protein band (7.2kd) completely produced by cutting as the representative of cutting effect, and the band of TEVp (29.5kDa) represents the promoter strength of TEVp. From the grayscale of the TEVp and monomer protein bands, we find out the cutting effect of the strongest promoter J23111 was weak than the second strong promoter J23106. It’s also supporting the results of our model that changing the strength of promoters can change the cutting effect and the concentration of single effectors in extracellular, providing theoretical support for the practical application of our project.

Fig.3 Cleavage effect under three promoters of TEVp.

Lane 1: protein purified from supernatant of bacteria liquid expressing TEVp with promoter J23110.

Lane 2: protein purified from supernatant of bacteria liquid expressing TEVp with promoter J23111.

Lane 3: protein purified from supernatant of bacteria liquid expressing TEVp with promoter J23106.

The mass spectrometry analysis (Fig.4) shows that His-A, the smallest component of the fusion protein, was detected.

Integration with model

Our model firstly ensured the most reasonable quantity proportion of TEV proteases and anti-Plasmodium fusion proteins. Then we supposed different secretion speed levels, obtained the most suitable combination of promoters in each condition. What surprised us is, one of these assumptions fitted our experiment well.

To see more details: NAU-CHINA-2019-wiki

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 2232
  Illegal NheI site found at 2255
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 444
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2582
  Illegal SapI.rc site found at 2930