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Team:Cambridge/Experiments/Plasmid Constructs
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=Constructs= | =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. | + | 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== | ==Expression Constructs== | ||
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===GA1=== | ===GA1=== | ||
Reflectin A1 is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies). | Reflectin A1 is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies). | ||
| - | [[File:cam_igem_map_GA1. | + | [[File:cam_igem_map_GA1.png| 700px]] |
===GA2=== | ===GA2=== | ||
Reflectin A1 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies). | Reflectin A1 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies). | ||
| - | [[File:cam_igem_map_GA2. | + | [[File:cam_igem_map_GA2.png | 700px]] |
===GA3=== | ===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). | 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). | ||
| - | [[File:cam_igem_map_GA3. | + | [[File:cam_igem_map_GA3.png | 700px]] |
===GA4=== | ===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). | 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). | ||
| - | [[File:cam_igem_map_GA4. | + | [[File:cam_igem_map_GA4.png | 700px]] |
===GA5=== | ===GA5=== | ||
Reflectin A2 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies). | Reflectin A2 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies). | ||
| - | [[File:cam_igem_map_GA5. | + | [[File:cam_igem_map_GA5.png | 700px]] |
===GA6=== | ===GA6=== | ||
Reflectin 1B is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies). | Reflectin 1B is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies). | ||
| - | [[File:cam_igem_map_GA6. | + | [[File:cam_igem_map_GA6.png | 700px]] |
| Line 37: | Line 39: | ||
===GA13=== | ===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). | 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). | ||
| - | [[File:cam_igem_map_GA13. | + | [[File:cam_igem_map_GA13.png | 700px]] |
===GA14=== | ===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). | 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). | ||
| - | [[File:cam_igem_map_GA14. | + | [[File:cam_igem_map_GA14.png | 700px]] |
| Line 48: | Line 50: | ||
===GA17=== | ===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. | 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. | ||
| - | [[File:cam_igem_map_GA17. | + | [[File:cam_igem_map_GA17.png |center | 700px ]] |
===GA18=== | ===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. | 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. | ||
| - | [[File:cam_igem_map_GA18. | + | [[File:cam_igem_map_GA18.png |center | 700px ]] |
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}} | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}} | ||
Latest revision as of 03:30, 22 September 2011
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).
GA2
Reflectin A1 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).
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).
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).
GA5
Reflectin A2 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).
GA6
Reflectin 1B is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).
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).
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).
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.
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.
