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Team:Edinburgh/BioBricks
From 2010.igem.org
BioBricks: Genomic BRIDGEs
For the BRIDGE protocol we have one main BioBrick, which is the construct required for the two-step markerless insertion, a composite construct of cat (chloramphenicol resistance) and sacB (prevention of growth on sucrose).
In addition, we submitted the up- and downstream sequences of an example of a useful gene to be removed from the E. coli genome using the BRIDGE protocol: tnaA, which produces indole, such that when it is 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 of various emission / absorbance wavelengths developed in the FORTH light communication framework are listed below. A few are resubmissions of existing parts (noted as such in their descriptions), whilst others are composite devices with promoters and reporter systems.
The BioBricks for red light production are two variants of mutated firefly luciferase (S284T and 356K) and their composite reporter systems.
| 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 corrected variants of 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 a modified version of 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 updated 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 a LovTAP reporter system based on work by Lausanne 2009.
| Name | Type | Link | Description |
|---|---|---|---|
| LovTAP and reporter system (RFP) | Composite | BBa_K322999 | Blue light sensor with RFP reporter system. |
The BioBricks for the green light sensing are the CcaS-PhoR light sensor and the associated parts necessary to create the desired reporter system.
| Name | Type | Link | Description |
|---|---|---|---|
| phoA promoter | Regulatory | BBa_K322115 | Promoter activated by the CcaS-PhoR pathway. |
| phoA promoter with lacZ | Composite | BBa_K322117 | phoA promoter coupled to lacZ reporter. |
| CcaS-PhoR | Composite | BBa_K322119 | Green light sensor. |
| CcaS-PhoR and reporter system (lacZ) | Composite | BBa_K322120 | Green light sensor with lacZ reporter system. |
