Protein Expression Vectors

As part of the protein expression process in the gram positive theraputics portion

Vector Design

f1 origin

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.

Lac I

Promoters

The expression vectors promoter are available in constitutive and inducible variety. The [http://partsregistry.org/Part:BBa_J23100 Constitutive promoters] are BBa J23100, J23113, and J23114. Inducible vectors include [http://partsregistry.org/Part:BBa_R0011 R0011] and T7, both of which are repressed by the Lac I protein.

Ribosome Binding Site

The Elowitz standard RBS [http://partsregistry.org/Part:BBa_B0034 B0034] is used on all vectors.

Building the Vectors

overview of what was done in one paragraph... links to protocols used (need to put up pcr (taq/fusion) digestion, ligation, transformation links to registry)

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Testing the Vectors

Protien Expression

The vector cassette was placed in 4 different plasmid backbone from the registry [http://partsregistry.org/Part:pSB1C3 pSB1C3], [http://partsregistry.org/Part:pSB1A3 pSB1A3], [http://partsregistry.org/Part:pSB3K3, pSB3K3], [http://partsregistry.org/Part:pSB4A5 pSB4A5] and [http://partsregistry.org/Part:BBa_E0040 GFP] was placed in the protein expression area of the vector. Data was pulled and expressed below....

f1 origin

The f1 origin was tested by comparing SS DNA harvest using part of the Kunkel’s mutagenesis (link) from CJ236 cells 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. The results are pictured below.