Team:Cambridge/Experiments/Plasmid Constructs

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=Constructs=
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As part of our project we designed a library of plasmid constructs for protein expression. Diagrams of all of them are give below.   
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As part of our project we designed a library of reflectin expression plasmids, as well as constructs with reflectin transcriptional fusions with GFP, and finally plasmids with reflectin targetted for export to the periplasm. Diagrams of all of them are given below.
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Plasmids containing synthetic versions of reflectins A1 and A2 (from ''Loligo'') and 1B (from ''Euprymna'') were kindly sent to us by Dr Wendy Crookes-Goodson.
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==Expression Constructs==
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===GA1===
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Reflectin A1 is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies).
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[[File:cam_igem_map_GA1.png| 700px]]
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===GA2===
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Reflectin A1 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).
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[[File:cam_igem_map_GA2.png | 700px]]
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===GA3===
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Reflectin A1 with a HIS tag at the N-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies).
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[[File:cam_igem_map_GA3.png | 700px]]
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===GA4===
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Reflectin A1 with a HIS tag at the N-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).
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[[File:cam_igem_map_GA4.png | 700px]]
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===GA5===
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Reflectin A2 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).
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[[File:cam_igem_map_GA5.png | 700px]]
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===GA6===
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Reflectin 1B is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).
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[[File:cam_igem_map_GA6.png | 700px]]
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==GFP fusions==
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===GA13===
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Reflectin A1 fused to GFP at the C-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies).
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[[File:cam_igem_map_GA13.png | 700px]]
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===GA14===
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Reflectin A1 fused to GFP at the C-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).
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[[File:cam_igem_map_GA14.png | 700px]]
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==Export Constructs==
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===GA17===
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Reflectin A1 is fused to the TorA signal peptide at the N-terminus, and his-tagged at the C-terminus. It is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies). TorA signal targets the protein to the Tat export pathway in E.coli.
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[[File:cam_igem_map_GA17.png |center | 700px ]]
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===GA18===
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Reflectin A1 is fused to the TorA signal peptide at the N-terminus, and fused to GFP at the C-terminus. It is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies). TorA signal targets the protein to the Tat export pathway in E.coli.
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[[File:cam_igem_map_GA18.png |center | 700px ]]
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Latest revision as of 03:30, 22 September 2011

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Contents

Constructs

As part of our project we designed a library of reflectin expression plasmids, as well as constructs with reflectin transcriptional fusions with GFP, and finally plasmids with reflectin targetted for export to the periplasm. Diagrams of all of them are given below.

Plasmids containing synthetic versions of reflectins A1 and A2 (from Loligo) and 1B (from Euprymna) were kindly sent to us by Dr Wendy Crookes-Goodson.

Expression Constructs

GA1

Reflectin A1 is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies). Cam igem map GA1.png

GA2

Reflectin A1 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies). Cam igem map GA2.png

GA3

Reflectin A1 with a HIS tag at the N-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies). Cam igem map GA3.png

GA4

Reflectin A1 with a HIS tag at the N-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies). Cam igem map GA4.png

GA5

Reflectin A2 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies). Cam igem map GA5.png

GA6

Reflectin 1B is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies). Cam igem map GA6.png


GFP fusions

GA13

Reflectin A1 fused to GFP at the C-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies). Cam igem map GA13.png

GA14

Reflectin A1 fused to GFP at the C-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies). Cam igem map GA14.png


Export Constructs

GA17

Reflectin A1 is fused to the TorA signal peptide at the N-terminus, and his-tagged at the C-terminus. It is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies). TorA signal targets the protein to the Tat export pathway in E.coli.

Cam igem map GA17.png

GA18

Reflectin A1 is fused to the TorA signal peptide at the N-terminus, and fused to GFP at the C-terminus. It is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies). TorA signal targets the protein to the Tat export pathway in E.coli.

Cam igem map GA18.png


Retrieved from "http://2011.igem.org/Team:Cambridge/Experiments/Plasmid_Constructs"