Difference between revisions of "Part:BBa K2623015"

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====Identification====
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===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.<br>
 
In our circuit of the build process, we have been doing nucleic acid gel electrophoresis to verify. After the loop is complete, sequencing verification.<br>
 
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[[Image:SAHS_Gel_2.png|thumb|400px|Fig.2]]</th><th></table>
 
[[Image:SAHS_Gel_2.png|thumb|400px|Fig.2]]</th><th></table>
 
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After the fluorescence identification, we performed a small amount of protein expression by SDS-PAGE.<br>
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====Verify the expression of SAHS protein====
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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.<br>
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https://static.igem.org/mediawiki/parts/4/49/SAHS_pro_Gel_1.png<br>
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SAHS is an exogenous protein with a signal peptide in front of it that allows the protein to be secreted outside the membrane. But our site organism is E. coli, because there is a cell wall, so the protein can not be secreted out of the membrane. Therefore, we can hardly see our target protein in the supernatant. Therefore, we can only obtain the protein we need from the cell pellet. After that, we connected our SAHS protein to the pET-28a plasmid, induced expression, and obtained a large amount of our target protein by ultrasonic disruption of the bacterial cell pellet.<br>
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[[Image:BBa K2623016.jpg|thumb|400px|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.]]</th><th>
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[[Image:BBa K2623015.jpg|thumb|400px|Fig.6 ]]</th><th></table>
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More information about our project can be found on our results page.http://2018.igem.org/Team:XMU-China/Results
 
More information about our project can be found on our results page.http://2018.igem.org/Team:XMU-China/Results
  

Revision as of 13:36, 17 October 2018


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.

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.
Fig.6



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