Team:NOVA LxPortugal/Parts
Parts
The strategy of our project foresees the validation of the selected alpha-pinene inducible promoters to activate the production of our nematicidal compound, spectinabilin, as a response to the higher levels of alpha-pinene in the pine tree after the nematode infection. To achieve this, we have designed different basic and composite parts. An already existing reporter gene part (BBa_K208000) containing an improved version of the Green Fluorescent Protein (GFP) was used to build the composite parts necessary to validate the selected promoters. In the composite parts, the expression of the reporter gene is controlled by potential alpha-pinene inducible promoters, these parts also include a terminator sequence in the downstream region. We have registered three different parts corresponding to the two different promoters tested as well as to the terminator.
Basic parts
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K3675000 | Regulatory | Alpha-pinene oxide lyase promoter from Pseudomonas rhodesiae. Regulates an enzyme involved in the first step of the alpha-pinene catabolic pathway . An RBS sequence was inserted after the annotated promoter sequence. | 54 bp |
| BBa_K3675001 | Regulatory | Enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase promoter from Pseudomonas putida KT2440. Regulates an enzyme involved in the alpha-pinene catabolic pathway. An RBS sequence was inserted after the annotated promoter sequence. | 666 bp |
| BBa_K3675002 | Reporter | This part encodes an enhanced Green Fluorescent Protein (GFPuv). This synthetic gene codes for a powerful reporter molecule for monitoring gene expression, protein localization and protein-protein interaction. An already existing part (BBa_K208000) contains this sequence. | 720 bp |
| BBa_K3675003 | Terminator | A terminator suitable for the construction of complex synthetic circuits capable of stopping the gene transcription. The sequence was demonstrated to be a strong terminator in Pseudomonas putida KT2440 but possibly can actuate in a variety of prokaryotic hosts | 158 bp |
Composite parts
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K3675004 | Composite | This composite part has an improved GFP coding gene (BBa_K3675002) with its expression controlled by a Pseudomonas rhodesiae regulatory region (BBa_K3675000) upstream. A strong terminator is inserted downstream to ensure that there is no leaky signal (BBa_K3675003) This part can be therefore used to detect the presence of alpha-pinene in the medium when transformed in a Pseudomonas putida strain capable of degrading alpha-pinene. | 946 bp |
| BBa_K3675005 | Composite | This composite part has an improved GFP coding gene (BBa_K3675002) with its expression controlled by a Pseudomonas putida regulatory region (BBa_K3675001) upstream. A strong terminator is inserted downstream to ensure that there is no leaky signal (BBa_K3675003) This part can be therefore used to detect the presence of alpha-pinene in medium when transformed in a Pseudomonas putida strain capable of degrading alpha-pinene. | 1558 bp |
