Team:EPFL/Parts Overview

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    Monomer and dimer design
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    Showing yeast captures copper
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Parts Overview

Contents

• Plasmid
• CUP1
• CUP1 dimers
• References

Plasmid

We have used the plasmid backbone pCTcon2-V51 to clone our DNA sequences. It was kindly given to us by the Laboratory of Protein Design and Immunoengineering of Professor Bruno Correia. This plasmid contains:

• Two selection markers:

  • An ampicillin (Amp+) resistance gene for genetic selection of transformed E. coli. Selection is achieved in a medium supplemented with ampicillin antibiotic.

  • A TRP gene as an auxotrophic marker for genetic selection of transformed trp- yeasts, unable to synthesize tryptophan. Selection is achieved in a medium lacking tryptophan.

• An inducible expression system composed of:

  • A Gal1 promoter allowing the transcription of our gene of interest only in the presence of galactose.

  • The yeast surface display:

    • The Aga2 coding sequence; the A-agglutinin-binding subunit Aga2 attaches to the yeast cell wall through disulfide bonds to Aga1p, a yeast endogenous protein.

    • A multiple cloning site, where we inserted our gene sequence by digesting the plasmid with the restriction enzymes NheI and BamHI.

    • The V5 tag to check expression either by immunoblot or immunocytochemistry.

Figure 1Scheme of the plasmid with a dimer inserted.

CUP1

In order to make the yeast express CUP1 on its outer membrane, we cloned the genomic CUP1 sequence into the plasmid pCTcon2V5 so that it would be linked to the Aga2 protein C-terminal. The part BBa_K4035001 is the DNA sequence composed of Aga2, CUP1 and V5 tag.

We wanted an inducible system, which could be activated only under a certain condition. In the plasmid, a Gal1 promoter is present upstream of the coding sequence. Our yeast surface display could thus be expressed only in the presence of galactose. This system is represented by the part BBa_K4035000.

CUP1 dimers

In order to improve our first version of the CUP1 extracellular expression in yeast, we have designed multiple dimers, namely two CUP1 proteins joined together with a linker. We have seven different dimers, each of them using a particular linker. For further information about the design of these dimers, see the Design page.

• Part K4035002 has a flexible linker made of 3 times the GGGGS amino acid sequence.

• Part K4035003 has a flexible linker made of 4 times the GGGGS amino acid sequence.

• Part K4035004 has a rigid linker made of 7 times the AP amino acid sequence.

• Part K4035005 has a rigid linker made of 3 times the EAAAK amino acid sequence.

• Part K4035006 has a rigid linker made of 4 times the EAAAK amino acid sequence.

• Part K4035007 has a semi-rigid linker made of the flexible GGGGS amino acid sequence followed by the EAAAK followed by a second GGGGS sequence.

• Part K4035008 has a semi-rigid linker made of the flexible GGGGS amino acid sequence followed by two times the EAAAK followed by a second GGGGS sequence.

The parts numbers BBa_K4035009 to BBa_K4035015 depict the extracellular expression system of these dimers, namely, the Gal1 promoter, the Aga2 protein and the dimer sequence followed by the V5 tag.

References

1. Chao, Lau, Hackel, Sazinsky, Lippow & Wittrup (2006)
   Isolating and engineering human antibodies using yeast surface display
   Nature Protocols, vol. 1, no. 2, pp. 755-768
   CrossrefGoogle Scholar

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