Team:NAU-CHINA/Part Collection
Part Collection
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Part Collection
This year, NAU-CHINA designed mainly 3 parts as a collection. By using different combinations of promoters with both TEV protease (BBa_K3139012) and Anti-Plasmodium fusion protein (BBa_K3139013), we explored the best combination of their expression strength, which led the least waste of TEV protease and the least pre-fusion protein with incomplete cleavage.
| Part Number | Name | Type |
|---|---|---|
| BBa_K3139014 | J23100-fusion protein-B0015-J23106-TEVp-TEVp site-HasA-B0010 | Composite |
| BBa_K3139015 | J23100-fusion protein-B0015-J23110-TEVp-TEVp site-HasA-B0010 | Composite |
| BBa_K3139016 | J23100-fusion protein-B0015-J23111-TEVp-TEVp site-HasA-B0010 | Composite |
Background
In our design, we described an anti-Plasmodium system with TEV protease cleavage system, anti-Plasmodium fusion protein system and the HasA secretion system. In the anti-Plasmodium system of NAU-2019, 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.
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.
Characterization
We constructed 3 recombinant plasmids. To obtain the best cleavage efficiency, we planned to replace the promoter of TEVp, observing the change of cleavage efficiency, and finally chose the most suitable promoter. We selected three promoters: J23111，J23110 and J23106, from the family of constitutive promoter parts, to construct 3 plasmids. We transformed these three plasmids into S. marcescens 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.
Results
The WB result shows several bands. 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.
Figure.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 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: PES model - RESULTS & ANALYSIS