Difference between revisions of "Part:BBa K2623015"

Revision as of 10:02, 7 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.

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

After the fluorescence identification, we performed a small amount of protein expression by SDS-PAGE.


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.
More information about our project can be found on our results page.http://2018.igem.org/Team:XMU-China/Results

Sequence and Features