Composite
SAHS 1

Part:BBa_K2623015

Designed by: Jun Dai   Group: iGEM18_XMU-China   (2018-09-28)
Revision as of 13:40, 17 October 2018 by Bb1984 (Talk | contribs) (→‎Verify the expression of SAHS protein)


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.

CAHS_Fig1.png

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
Fig.2


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.
BBa K2623015.jpg

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.












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


[edit]
Categories
Parameters
None