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From 2013.igem.org

Pet-mCherry is a β-barrel autotransporter with a mCherry passenger.

Contents 1 Motivation 2 Design 3 Assembly 4 Characterization 4.1 Is the LacI operator working? 4.2 Is mCherry on the outside of the cell? 5 References

Motivation

In 2012 the Georgia Tech iGEM team developed a novel biosensor based off of green fluorescent protein. The sensor consisted of two subunits of the protein that separately were inactive but once dimerized expressed fluorescence. From this project, we began thinking about how we could develop more complex sensing technology in bacteria. Taking into consideration how mammalian cells sense and react to their environment, we started asking the question: Can bacteria express human integrins?

To start answering this question, we needed to find a way to transport large proteins and anchor them to the outside of the cell. Autodisplay technology seemed like one possible solution to this problem.

Design

To comply with BioBrick standard 10, Phe-76 was re-optimized to remove a EcoRI site withing the part. A T7 promoter with a lacI operator was added with the RBS (AGGA) to the PET_mCherry. A standard assembly 10 prefix and suffix was then added. In future work, other autodisplay technologies are hoped to be tested to express a number of proteins. [http://www.uniprot.org/uniprot/O68900 Uniprot]

Primers:
ig12 SP/Pet_mCherry_b1/0	AGTCAGGAATTCGCGGCCGCTTCTAGAGG
ig13 SP/Pet_mCherry_b2/469	CGTATGAAGGCACCCAGACCGCTAAACTGAAA
ig14 ASP/Pet_mCherry_b1/500	TTTCAGTTTAGCGGTCTGGGTGCCTTCATACG
ig15 SP/Pet_mCherry_b3/928	CGGGTGCTTACAACGTGAACATCAAACTGGAC
ig16 ASP/Pet_mCherry_b2/959	GTCCAGTTTGATGTTCACGTTGTAAGCACCCG
ig17 SP/Pet_mCherry_b4/1352	CAAACTGGAAGGTGCGAACAACCTGCTGC
ig18 ASP/Pet_mCherry_b3/1380	GCAGCAGGTTGTTCGCACCTTCCAGTTTG
ig19 SP/Pet_mCherry_b5/1816	TGTTCACCGGTGTTACCATGACCTACACCGAC
ig20 ASP/Pet_mCherry_b4/1847	GTCGGTGTAGGTCATGGTAACACCGGTGAACA
ig21 SP/Pet_mCherry_b6/2265	GGTTACCAGTTCGACCTGTTCGCTAACGGTGA
ig22 ASP/Pet_mCherry_b5/2296	TCACCGTTAGCGAACAGGTCGAACTGGTAACC
ig23 ASP/Pet_mCherry_b6/2516	ACTCTGCAGCGGCCGCTACTAGTATTATTATC

Assembly

The 6 "Gblocks" that we synthesized using IDT were assembled using overlap extension PCR.

Characterization

The cells for the colonies BEP1 and BEP3 were characterized using flow cytometry. Our goals for this characterization were to show activity of the LacI operator and confirm the expression of mCherry on the outside of the cell. Cells expressing H6, a single strand antibody that favors fibrin, were used as a negative control. Flow cytometry data for PET-mCherry and H6 negative controlData was gated to capture the population of interest for the control and BEP3. The increase in events outside of the gate post-induction withIPTG for the control indicates cell induced death due to over-expression. Increased expression of mCherry after induction does not induce cytotoxicity.

Is the LacI operator working?

Is mCherry on the outside of the cell?

References

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