Module 1
Salicylic acid production and SABP2 expression
| Part Name | Genetic Construct / Composition | Type | Description |
|---|---|---|---|
| Nt_SABP2 |
[prefix]
Anderson promoter
Standard RBS
(BBa_B0034) SABP2 CDS with stop codon TAA Standard iGEM double terminator (BBa_B0015) [suffix] |
Composite | Salicylic Acid Binding Protein 2 is an esterase that converts methyl salicylate into salicylic acid. This BioBrick uses an Anderson promoter for constant expression of the esterase. It will be used in conjunction with other DNA parts so that the esterase is constantly expressed and ready for any MeSA that enters the cell. The gene is native to Nicotiana tabacum and other tobacco plants. |
| Nt_SABP2_lac |
[prefix]
T7 promoter + lac operator
Standard RBS
(BBa_B0034) SABP2 CDS with stop codon TAA Standard iGEM double terminator (BBa_B0015) [suffix] |
Composite | Salicylic Acid Binding Protein 2 is an esterase that converts methyl salicylate into salicylic acid. This version uses a T7 promoter for selective induction with IPTG and is intended for use in BL21(DE3) cells. T7 is used to produce as much gene product as possible. The gene is native to Nicotiana tabacum and other tobacco plants. |
Module 2
Salicylate-responsive transcription and fluorescence
| Part Name | Genetic Construct / Composition | Type | Description |
|---|---|---|---|
| Pp_NahR |
[prefix]
Lac UV5 promoter + lac operator
Standard RBS
(BBa_B0034) NahR CDS with stop codon TAA Standard iGEM double terminator (BBa_B0015) [suffix] |
Composite | Salicylate-dependent activator protein that binds to the PSal promoter. When salicylic acid is not present, the transcription factor does not bind to RNA polymerase. When salicylic acid is bound, it binds to RNA polymerase and activates transcription of the downstream gene. NahR production is controlled by the Lac UV5 promoter, which is induced by IPTG. The gene is native to Pseudomonas putida. |
| Pp_SalR |
[prefix]
Lac UV5 promoter + lac operator
Standard RBS
(BBa_B0034) SalR CDS with stop codon TAA Standard iGEM double terminator (BBa_B0015) [suffix] |
Composite | Salicylate-dependent repressor protein that binds to the PSal promoter. Unlike NahR, it acts as a repressor when not bound to salicylic acid, actively deterring transcription. When bound to salicylic acid, it activates the gene by binding to RNA polymerase. SalR production is controlled by the Lac UV5 promoter, which is induced by IPTG. The gene is native to Pseudomonas putida. |
| msGFP2_SA |
[prefix]
PSal promoter
Standard RBS
(BBa_B0034) msGFPII CDS with stop codon TAA Standard iGEM double terminator (BBa_B0015) [suffix] |
Composite | Monomeric superfolder Green Fluorescent Protein 2 is a modified version of GFP. On its own, this BioBrick cannot express GFP and requires a transcription factor. It will be used with another gene so that induction allows msGFP expression, enabling fluorescent assays to calculate the amount of gene product produced. The original gene is native to Aequorea victoria. |
Module 3
Engineering the salicylic acid biosynthetic pathway
| Part Name | Genetic Construct / Composition | Type | Description |
|---|---|---|---|
| EntC |
[prefix]
BBa_J23101 promoter (Anderson)
Standard RBS
(BBa_B0034) EntC CDS with stop codon TAA Standard iGEM double terminator (BBa_B0015) [suffix] |
Composite | Isochorismate synthase is an isomerase that converts chorismate to isochorismate. It is part of the Shikimate pathway that converts 2-phosphoenolpyruvate into salicylic acid. E. coli does not naturally have the last two genes required to make salicylic acid and stops at chorismate. This gene is introduced into E. coli to help finish the pathway. The gene is native to Escherichia coli. |
| Pa_PchB |
[prefix]
PRha promoter
Standard RBS
(BBa_B0034) PchB CDS with stop codon TGA L3S2P21 terminator standard intrinsic terminator [suffix] |
Composite | Isochorismate pyruvate lyase is a lyase that cleaves isochorismate into salicylate and pyruvate. It is part of the Shikimate pathway that converts 2-phosphoenolpyruvate into salicylic acid. E. coli does not naturally have the last two genes required to make salicylic acid and stops at chorismate. This gene is introduced to finish the pathway and allow E. coli to make salicylic acid and pyruvate. The gene is native to Pseudomonas aeruginosa. |
Module 4
Salicylic acid-responsive insecticidal protein expression
| Part Name | Genetic Construct / Composition | Type | Description |
|---|---|---|---|
| Bt_Vip3A_lac |
[prefix]
T7 promoter + lac operator
Standard RBS
(BBa_B0034) Vip3A CDS with stop codon TAA Standard iGEM double terminator (BBa_B0015) [suffix] |
Composite | Vegetative insecticidal protein 3A is an insecticidal protein protoxin that is cleaved into an active toxin by strong alkaline proteases present in the insect midgut. The toxin binds to receptors on the gut interior and has a long, needle-like extension that pierces the gut lining, making it porous. This gene uses a T7 promoter for maximal protein expression and protein analysis. The gene is native to Bacillus thuringiensis. |
| Bt_Vip3A_SA |
[prefix]
PSal promoter
Standard RBS
(BBa_B0034) Vip3A CDS with stop codon TAA Standard iGEM double terminator (BBa_B0015) [suffix] |
Composite | Vegetative insecticidal protein 3A is an insecticidal protein protoxin that is cleaved into an active toxin by strong alkaline proteases present in the insect midgut. This Vip3A gene is under a PSal promoter, allowing it to be responsive to salicylic acid. The gene cannot be transcribed on its own and can be transcribed whenever there is a transcription factor associated with the gene. The gene is native to Bacillus thuringiensis. |
Module 5
Theophylline-responsive regulation and toxin-antitoxin control
| Part Name | Genetic Construct / Composition | Type | Description |
|---|---|---|---|
| msGFP2_theo |
[prefix]
BBa_J23101 promoter (Anderson)
Theophylline aptamer in 5′ UTR
covering the RBS Standard RBS (BBa_B0034) msGFPII CDS with stop codon TAA Standard iGEM double terminator (BBa_B0015) [suffix] |
Composite | Monomeric superfolder Green Fluorescent Protein 2 is a modified version of GFP. The original gene is native to Aequorea victoria. This GFP is under a theophylline riboswitch. When theophylline binds to the riboswitch, it opens the hairpin formed by the aptamer mRNA, revealing the RBS and inducing translation. The riboswitch was synthetically designed and is not indigenous to any organism. |
| RelE |
[prefix]
BBa_J23114 promoter
(Anderson / weak) Standard RBS (BBa_B0034) RelE CDS with stop codon TGA Standard iGEM double terminator (BBa_B0015) [suffix] |
Composite | RelE is a toxic mRNA interferase that cleaves mRNA at the A-site in ribosomes. The gene is native to Escherichia coli. |
| RelB |
[prefix]
BBa_J23100 promoter (Anderson)
Theophylline riboswitch in 5′ UTR
with standard RBS (BBa_B0034) RelB CDS with stop codon TGA Standard iGEM double terminator (BBa_B0015) [suffix] |
Composite | RelB is a ribbon-helix-helix repressor that forms dimers, preventing transcription of RelE and counteracting the RelE protein itself. The gene is native to Escherichia coli. Theophylline, when bound to the riboswitch, opens the hairpin formed by the mRNA and reveals the RBS to induce translation. The riboswitch was synthetically designed and is not indigenous to any organism. |