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.
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. |
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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.
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.
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）.
More information about our project can be found on our results page.http://2018.igem.org/Team:XMU-China/Results
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 279
Illegal XhoI site found at 503 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1152
Illegal AgeI site found at 1264 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 558
Illegal SapI.rc site found at 553
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