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Team:Edinburgh/BioBricks
From 2010.igem.org
BioBricks: Genomic BRIDGEs
For this part of the project we will have one main BioBrick, which will be the construct required for the two-step markerless insertion, encompassing cat (chloramphenicol resistance) and sacB (prevents growth on sucrose).
Chloramphenicol resistance has already been well characterised in the registry, so our characterisation has mainly focused on sacB.
In addition we will probably be submitting the construct with the up- and down- stream sequences of useful genes which can be removed, e.g. tnaA, which produces indole - when removed E. coli no longer smells!
| Name | Type | Link | Description |
|---|---|---|---|
| cat | Translational Unit | BBa_K322210 | Chloramphenicol acetyltransferase (chloramphenicol resistance gene). |
| sacB | Translational Unit | BBa_K322921 | Bacillus subtilis levansucrase, lethal to E. coli in the presence of sucrose. |
| cat-sacB construct | Composite | BBa_K322922 | Construct necessary for BRIDGE protocol. |
| tnaA upstream region | Other | BBa_K322705 | Upstream region of E. coli tryptophanase locus, used for targetting genes to this locus using BRIDGE. |
| tnaA downstream region | Other | BBa_K322706 | Downstream region of E. coli tryptophanase locus, used for targetting genes to this locus using BRIDGE. |
BioBricks: Bacterial BRIDGEs
The light-sensing and light-production BioBricks used in the bacterial BRIDGEs component of the project are listed below. A few are resubmissions of existing parts (noted in their description), whilst others are composite devices with promoters and reporter systems.
The BioBricks for red light production are two variants of mutated firefly luciferase.
| Name | Type | Link | Description |
|---|---|---|---|
| Firefly luciferase S284T mutant | Coding | BBa_K322246 | Firefly luciferase from Photinus pyralis mutated towards the red spectrum. |
| S284T mutant luciferase under lac promoter | Composite | BBa_K322247 | Lac promoter followed by firefly luciferase S284T mutant. |
| Firefly luciferase 356K mutant | Coding | BBa_K322211 | Firefly luciferase S284T mutant from Photinus pyralis re-mutated towards the red spectrum. |
| 356K mutant luciferase under lac promoter | Composite | BBa_K322212 | Lac promoter followed by firefly luciferase 356K mutant. |
The BioBricks for blue light production are the LuxAB, LuxCDE, and LumP proteins from Edinburgh 2009 in various combinations, contained in new plasmids and with various reporter systems.
| Name | Type | Link | Description |
|---|---|---|---|
| luxAB | Translational Unit | BBa_K322139 | Bacterial luciferase producing blue light (from X. luminescens). |
| luxCDE | Composite | BBa_K322312 | Codes for a fatty acid reductase complex that makes the fatty acids necessary for LuxAB. |
| lumP | Translational Unit | BBa_K322007 | Lumazine protein to shift the wavelength of LuxAB to blue. |
| Name | Type | Link | Description |
|---|---|---|---|
| luxAB under lac promoter | Composite | BBa_K322140 | Lac promoter followed by luxAB. |
| luxAB and luxCDE under lac promoter | Composite | BBa_K322141 | Lac promoter followed by luxAB and luxCDE. |
| luxAB and lumP | Composite | BBa_K322149 | luxAB and lumP composite part. |
| luxAB and lumP under lac promoter | Composite | BBa_K322150 | Lac promoter followed by luxAB and lumP |
The BioBricks for green light production are the wildtype firefly luciferase from Ljubljana 2007 and a codon-optimised variant with brighter output.
| Name | Type | Link | Description |
|---|---|---|---|
| Firefly luciferase | Coding | BBa_K322237 | Ljubljana 2007's BBa_I712019 in pSB1C3, from Photinus pyralis. |
| Wildtype luciferase under lac promoter | Composite | BBa_K322238 | Lac promoter followed by wildtype luciferase. |
| Codon optimised bright firefly luciferase | Translational Unit | BBa_K322451 | Firefly luciferase from Photinus pyralis mutated for increased bioluminescence. |
The BioBricks for red light sensing are the Cph8 systems from UT Austin 2004 and Harvard 2008 in a variety of combinations, contained in a new plasmid and with two different reporter systems.
| Name | Type | Link | Description |
|---|---|---|---|
| Phycocyanobilin synthesis operon | Translational Unit | BBa_K322122 | Harvard 2008's BBa_K098010 in pSB1C3. |
| Phycocyanobilin synthesis operon without terminator | Translational Unit | BBa_K322123 | BBa_K098010 without terminator, allowing addition of cph8. |
| cph8 with RBS | Translational Unit | BBa_K322124 | Light sensing protein for red light (BBa_I15010 in pSB1C3). |
| cph8 and reporter system (lacZ) | Composite | BBa_K322125 | Red light sensor with lacZ reporter system. |
| cph8 and reporter system (EYFP) | Composite | BBa_K322126 | Red light sensor with EYFP reporter system. |
| Phycocyanobilin synthesis genes with cph8 | Composite | BBa_K322127 | The entire phycocyanobilin - cph8 system, without reporter system. |
| Phycocyanobilin synthesis genes with cph8 and EYFP reporter system | Composite | BBa_K322128 | The entire phycocyanobilin - cph8 system, with EYFP reporter system. |
The BioBrick for blue light sensing is the LovTAP reporter system from Lausanne 2009 in a new plasmid.
| Name | Type | Link | Description |
|---|---|---|---|
| LovTAP and reporter system (RFP) | Composite | BBa_K322999 | Blue light sensor with RFP reporter system. |
