Part:BBa_K2623015

Secretory-abundant heat soluble protein "SAHS " (promoter, RBS, RFP and double terminator)

Summary

TDP circuit is used to express exocrine protein SAHS BBa_K2623007. We added RFP BBa_E1010 as a reporter gene to the gene circle for us to screen and validate. Because our aim is to obtain a large number of SAHS proteins, the constant promoter J23100 is chosen to allow SAHS to be expressed continuously. The sequence encoding for the gene is preceded by a constant promoter BBa_J23100 and an RBS BBa_B0034, and followed by a double terminator BBa_B0015.

Identification

In our circuit of the build process, we have been doing nucleic acid gel electrophoresis to verify. After the loop is complete, sequencing verification.

Fig.1 The result of this plasmid cut with enzyme EcoRI and PstI.	Fig.2 The result of this plasmid cut with enzyme EcoRI and PstI.

Verify the expression of SAHS protein

Firstly, we used the biobick E1010 (mRFP) as our report gene to make sure the circuit for expressing the SAHS protein was constructed precisely. But we did not observe the distinct color on the plate as it was so weak unless the E.coli was cultured in a tube and centrifuged. So we had a new test by using the biobick K592009（blue chromoprotein）as our report gene and got a distinct color on the plate.

Fig.3 These are the pictures our fluorescently characterized plate and bacterial pellets. The report gene on the left is K592009, and the one on the right is E1010. Some colonies on the left are blue, which are the DH5α that we successfully transferred to the designed genetic loop. The leftmost EP tube in the right photo is the control, and the other two EP tubes contain DH5α which we had successfully transferred to the designed genetic loop.

And then, we transformed the plasmid to the E.coli(BL21), which is always used to express proteins with high efficience to verify the SAHS protein was produced successfully with a small scale.Besides, whether the SAHS protein with a signal peptide could be secreted was determined by SDS-PAGE.

Fig.4 The marker is on the lest, followed by our control group ( the BL21 with the empty plasmid), and the third well is the concentrated supernatant. The unit of the marker is "Kd"

As shown in the image above, We also explored the appropriate temperature for SAHS protein expression. As we expected, protein expression was be more efficient at 30 °C. However, we did not find the SAHS protein band in the supernatant even when it was concentrated by ultrafiltration. So we speculated that cell wall obstructed the secretion of protein on the basis of 2016 Peking University’s working.(see more information on http://2016.igem.org/Team:Peking/Secretion)

Therefore, we have to design a new protein purification program. To get enough protein produces for the following experiment, the gene was cloned into the vector pET-28a with high expression lever combined with E.coli(BL21). Besides, Two HIS-TAGs on the end of N-and C-terminal was produced and allow SAHS protein to bind with Nickel column(like Ni-NTA) for purification. Meanwhile, we purifiedthe sample with heat water bath considering the characterization of heat stability by following the reference.

Fig.5 The picture on the left is a series of temperature gradient processed protein samples. The number represents the temperature (°C). And the picture on the right is the gel map of our final purified high purity SAHSprotein.

So, we designed a set of temperature gradients from 70°C to 90°C, to explore the appropriate condition. What’s more, we chose the 85℃ for 15mins finally for large scale purification. But given that there were many other protein bands, we combined heating with Nickel column for producing high quality protein. Finally, we tested two patterns, heating-Ni-NTA and Ni-NTA-heating, and found the later is better.

After that, we obtained a protein sample with high concentration and purity by using this protein purification method（figure 5）.

Verify the role of SAHS protein in preserving biological activity

In order to verify the preservation effect of SAHS protein, we the lyophilization method. First, we tested the concentration of purified SAHS protein, which ranged from 0.3 g/L to 0.6 g/L. Then, we concentrated the purified protein to control the protein concentration to about 1 g/L.

We selected Taq enzyme as the protein preserved in our experiment, using SAHS protein as protective agent, and set a series of concentration gradients: 1g/L, 0.5g/L, 0.1g/L, 0.01g/L and 0g/L， then stored with lyophilization. In order to verify the preservation effect of SAHS protein, we resuscited the lyophilized sample and performed PCR experiments to verify the activity of Taq enzyme. The activity of Taq enzyme can be reflected by the product of PCR.

Fig.7 PCR results of Taq enzymes preserved at different SAHS protein concentrations. Blank is the group without the Taq enzyme.

We selected different PCR systems for verification. We found that some short fragments were easy to succeed, but there was no obvious gradient. However, when we replaced them with some other fragments that are difficult to succeed or very long, there would show a very obvious gradient. We think it must be that a shorter fragments were too easy to succeed, and made the base too large. The two fragments above are pET-28a, which had a relatively obvious gradient and also had a good repeatability.
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Fig.8 The result of the comparison of PBS and ultra-pure water.Blank is the group that the Taq enzyme has not been lyophilized and without SAHS protein.

In addition, we also made a comparison between PBS and ultra-pure water. We found that regardless of the presence of TDP, the lyophilized system added with PBS could not make the correct band. On the contrary, there was only one large band which was much larger than 8000bp. We think it may be a PCR mismatch. Considering that PBS contained a large amount of salt, it was likely that these salt ions affected the binding of Taq enzyme to Mg2+.

Fig.9 The ruselt of the success rate of PCRwith Taq enzymes preserved at different SAHS protein concentrations.

Then, we tried some shorter fragments for PCR, and interfered with the PCR system by reducing time extension and reducing the concentration of the template in order to explore the success rate of PCR. As the figure showed, when the concentration of SAHS protein was 0.5g/L, the success rate of PCR was the highest. We suspected that excessive concentration of SAHS protein might affect the normal function of the Taq enzyme and might affect the binding of the enzyme to the substrate. This reminded us of the results of TUDelft in 2017. They also got the result of that 0.5g/L was better than 1g/L in some cases. Eventually, they took a concentration of 1g/L to preserve Cas13a. They discovered that while CAHS 94205 preserved Cas13a’s RNase-like activity after drying, CAHS 94205 could not preserve its specificity. This is exactly the same as ours when the concentration is 1g/L. Therefore, we believe that it is possible that they just did not find the most suitable storage concentration, and if the concentration is properly reduced, the results may be much better.

Conclusion

According to the experimental results of lyophilization above, we can clearly see the preservation effect of SAHS protein on protein activity. Secondly, when the concentration is too high, SAHS protein may affect the normal function of protein. Therefore, it is necessary to find the most appropriate concentration. The optimal concentration may vary slightly among different proteins, but the preservation of biological activity by SAHS proteins is undoubtable. We believe that SAHS protein is a promising bioactive protective agent for different preservative substances, as long as the appropriate preservation system is set up.
More information about our project can be found on our results page.http://2018.igem.org/Team:XMU-China/Results

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
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 279
  Illegal XhoI site found at 503
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1152
  Illegal AgeI site found at 1264
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 558
  Illegal SapI.rc site found at 553