Team:Washington/Tools Created/New Vectors

Protein Expression Vectors

Vector Design

The protein expression cassette is designed to go into any biobrick plasmid backbone. It was placed in 4 different plasmid backbone from the registry pSB1C3, pSB1A3, pSB3K3,and pSB4A5 by our lab.

The expression vector promoters are available in constitutive and inducible variety. The Constitutive promoters are BBa J23100 and J23114. Inducible vectors include R0011 and T7, both of which are repressed by the Lac I protein.

Lac I gene inserted to ensure regulation of the Lactose inducible promoters

Antibiotics resistance based on biobrick plasmid backbone used.

The Elowitz standard RBS B0034 is used on all vectors.

Origin of replication for the M13 series of phages. When included in plasmids that are infected by M13 helper phage will generate SS DNA of the plasmid.

Plasmid copy number based on biobrick plasmid backbone used.

Restriction cut sites based on biobrick standards.

Building the Vectors

The expression cassettes were built using the biobrick construction tutorials.

Testing the Vectors

Protein Expression

The vector cassette was placed in 4 different plasmid backbones from the registry pSB1C3, pSB1A3, pSB3K3, pSB4A5 and GFP was placed in the protein expression area of the vector. Data was pulled and expressed below....

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f1 origin

The f1 origin was tested by comparing SS DNA harvest using part of the Kunkel's mutagenesis. CJ236 cells were infected with M13K07 helper phage. The CJ236 cells varied in the present or absence of the f1 origin on the pSB1A3 or pSB3K3 plasmid. The SS DNA harvest was then run on a 50ml 1% agarose gel at 90V for 45minutes.