Our team has planned to create the following parts for our project, both level 0 parts, level 1 parts and level 2 parts. At the instance where we have successfully cloned the part and sequence verified the part, we have added the part sequence onto the Registry. At any instances where the Registry number is NA, it means unfortunately, we were unable to successfully made clone the device and sequence verify it.
Basic parts we incorporated into the Level 0 accepting vector (p18-Uac)
| Name | Type | Description | Length | Registry no. |
|---|---|---|---|---|
| P_BADmin | Promoter | Promoter induced by arabinose (activation) | 77 bp | BBa_K4491007 |
| PSigW | Promoter | Promoter induced by SigW (activation) | 36 bp | BBa_K4491103 |
| psinR | Promoter | Promoter repressed by sinR | 59 bp | BBa_K4491001 |
| mVenus | CDS | Yellow fluorescent protein | 717 bp | BBa_K4491104 |
| StayGold GFP | CDS | Green fluorescent protein | 654 bp | BBa_K4491105 |
| AS16 | CDS | Anti-sigma factor pair with ECF16 | 645 bp | BBa_K4491005 |
| AS20 | CDS | Anti-sigma pair with ECF20 | 432 bp | BBa_K4491107 |
| RSiW | CDS | Anti-sigma pair with SigW | 264 bp | BBa_K4491108 |
| tetR^DN | CDS | mutated version of tetR that binds to tetR and inactivates it | 615 bp | BBa_K4491109 |
| ECF20992 | CDS | Sigma factor that activtates p20_992 | 582 bp | BBa_K4491110 |
| SigW | CDS | Sigma factor that activates pSigW | 564 bp | BBa_K4491111 |
| sinR | CDS | Quorum-sensing molecule that represses psinR | 336 bp | BBa_K4491112 |
| DT5 | Terminator | ultra-strong double terminator to terminate transcription in both direction | 110 bp | BBa_K4491003 |
| DT54 | Terminator | ultra-strong double terminator to terminate transcription in both direction | 112 bp | BBa_K4491004 |
| DT3 | Terminator | ultra-strong double terminator to terminate transcription in both direction | 152 bp | BBa_K4491002 |
| L3S2P55 | Terminator | synthetic high-efficiency terminator | 61 bp | BBa_K4491113 |
| AraE | CDS | Arabinose transporter | 1419 bp | BBa_K4491114 |
| MegaT | RBS, CDS and Terminator | RBS, CDS and Terminator linked together as a part for use in JUMP assembly | 800 bp | BBa_K4491133 |
| MegaT_RFP | RBS, CDS and Terminator | RBS, CDS and Terminator linked together as a part for use in JUMP assembly with SapI site added for modulatiry | 1085 bp | BBa_K4491115 |
| afraGFP | CDS | Open Reporter from FreeGenes iGEM Distribution Kit 2 | 681 bp | BBa_K4491138 |
| efasGFP | CDS | Open Reporter from FreeGenes iGEM Distribution Kit 2 | 663 bp | BBa_K4491141 |
| Name | Type | Description | Registry no. |
|---|---|---|---|
| P_BAD AP1 | Promoter | Promoter induced by arabinose (activation) | BBa_K4491007 |
| P_BAD AP2 | Promoter | Promoter induced by arabinose (activation) | BBa_K4491007 |
| P_BAD AP3 | Promoter | Promoter induced by arabinose (activation) | BBa_K4491007 |
| P_BAD AP4 | Promoter | Promoter induced by arabinose (activation) | BBa_K4491007 |
| P_BAD AP5 | Promoter | Promoter induced by arabinose (activation) | BBa_K4491007 |
| P_BAD AP6 | Promoter | Promoter induced by arabinose (activation) | BBa_K4491007 |
| P_BAD AP7 | Promoter | Promoter induced by arabinose (activation) | BBa_K4491007 |
| P_BAD AP8 | Promoter | Promoter induced by arabinose (activation) | BBa_K4491007 |
| P_BAD AP9 | Promoter | Promoter induced by arabinose (activation) | BBa_K4491007 |
As an intermediate step to the full antithetic integral controller circuit, characterisation circuit and the open loop perturbation circuit, the following is made. Note that number 52 of the table refers to the transcription unit we inserted into the downstream site for the perturbation circuit. For details, please visit the Engineering Success page.
| Number | Function | Promoter | RBS | CDS | Terminator | Registry no. |
|---|---|---|---|---|---|---|
| 1 | Antithetic integral controller (ECF20, Module A) | p_20992 | B0032 | cinR^AM | MegaT | BBa_K4491116 |
| 2 | Antithetic integral controller (ECF20, Module A) | p_20992 | B0034 | cinR^AM | MegaT | BBa_K4491117 |
| 3 | Antithetic integral controller (ECF20, Module B) | J23106 | B0034 | ECF20 | DT3 | NA |
| 4 | Antithetic integral controller (ECF20, Module B) | J23105 | B0034 | ECF20 | DT3 | NA |
| 5 | Antithetic integral controller (ECF20, Module B) | J23100 | B0032 | ECF20 | DT3 | NA |
| 6 | Antithetic integral controller (ECF20, Module B) | J23106 | B0032 | ECF20 | DT3 | NA |
| 7 | Antithetic integral controller (ECF20, Module B) | J23105 | B0032 | ECF20 | DT3 | NA |
| 8 | Antithetic integral controller (ECF20, Module C) | P_Cin | B0032 | AS20 | DT5 | BBa_K4491118 |
| 9 | Antithetic integral controller (ECF20, Module C) | P_Cin | B0033 | AS20 | DT5 | BBa_K4491119 |
| 10 | Characterisation (ECF20, Module A) | p_20992 | B0032 | mVenus | DT5 | BBa_K4491120 |
| 11 | Characterisation (ECF20, Module B) | P_BADwt (from CIDAR MoClo) | B0032 | ECF20 | DT3 | NA |
| 12 | Characterisation (ECF20, Module C) | Ptet* | B0032 | AS20 | DT54 | BBa_K4491121 |
| 13 | Antithetic integral controller (sinR, Module A) | psinR | B0032 | vanR^AM | MegaT | BBa_K4491122 |
| 14 | Antithetic integral controller (sinR, Module A) | psinR | B0034 | vanR^AM | MegaT | NA |
| 15 | Antithetic integral controller (sinR, Module B) | J23100 | B0032 | SinR | DT3 | NA |
| 16 | Antithetic integral controller (sinR, Module B) | J23106 | B0032 | SinR | DT3 | NA |
| 17 | Antithetic integral controller (sinR, Module B) | J23100 | B0033 | SinR | DT3 | NA |
| 18 | Antithetic integral controller (sinR, Module B) | J23106 | B0033 | SinR | DT3 | BBa_K4491123 |
| 19 | Antithetic integral controller (sinR, Module C) | P_VanCC | B0034 | SinI | DT5 | NA |
| 20 | Antithetic integral controller (sinR, Module C) | P_VanCC | B0032 | SinI | DT5 | NA |
| 21 | Characterisation (sinR, Module A) | psinR | B0032 | mVenus | DT5 | BBa_K4491124 |
| 22 | Characterisation (sinR, Module B) | pBADwt (from CIDAR MoClo) | B0032 | sinR | DT3 | BBa_K4491125 |
| 23 | Characterisation (sinR, Module C) | Ptet* | B0032 | sinI | DT54 | BBa_K4491126 |
| 24 | Placeholder (Module D) | L3S2P55 | NA | NA | NA | BBa_K4491113 |
| 25 | Perturbation Circuit (Activation, Module D) | PTet* | B0032 | cinR^AM | MegaT | BBa_K4491127 |
| 26 | Perturbation Circuit (Repression, Module D) | PTet* | B0032 | vanR^AM | MegaT | BBa_K4491128 |
| 27 | Antithetic integral controller (ECF16, Module A) | p_163622 | B0032 | cinR^AM | MegaT | BBa_K4491129 |
| 28 | Antithetic integral controller (ECF16, Module A) | p_163622 | B0034 | cinR^AM | MegaT | BBa_K4491130 |
| 29 | Antithetic integral controller (ECF16, Module B) | J23106 | B0034 | ECF16 | DT3 | NA |
| 30 | Antithetic integral controller (ECF16, Module B) | J23105 | B0034 | ECF16 | DT3 | NA |
| 31 | Antithetic integral controller (ECF16, Module B) | J23100 | B0032 | ECF16 | DT3 | NA |
| 32 | Antithetic integral controller (ECF16, Module B) | J23106 | B0032 | ECF16 | DT3 | NA |
| 33 | Antithetic integral controller (ECF16, Module B) | J23105 | B0032 | ECF16 | DT3 | BBa_K4491131 |
| 34 | Antithetic integral controller (ECF16, Module C) | P_Cin | B0032 | AS16 | DT5 | BBa_K4491137 |
| 35 | Antithetic integral controller (ECF16, Module C) | P_Cin | B0033 | AS16 | DT5 | BBa_K4491140 |
| 36 | Characterisation (ECF16, Module A) | p_163622 | B0032 | mVenus | DT5 | BBa_K4491143 |
| 37 | Characterisation (ECF16, Module B) | P_BADwt (from CIDAR MoClo) | B0032 | ECF16 | DT3 | BBa_K4491145 |
| 38 | Characterisation (ECF16, Module C) | PTet* | B0032 | AS16 | DT54 | BBa_K4491144 |
| 48 | Open loop perturbation (ECF20, Module C) | PTet* | B0032 | ECF20 | DT5 | NA |
| 49 | Open loop perturbation (Module D) | J23106 | B0032 | tetR | DT54 | NA |
| 50 | Open loop perturbation (sinR, Module C) | PTet* | B0032 | sinR | DT5 | NA |
| 51 | Open loop perturbation (ECF16, Module C) | PTet* | B0032 | ECF16 | DT5 | NA |
| 52 | Perturbation (Downstream site) | J23106 | B0032 | tetR | ECK120029600 | BBa_K4491145 |
For the characterisation of StayGold GFP in the backbone pJUMP27-1A, the following Level 1 parts are made
| Number | Promoter | RBS | CDS | Terminator | Registry no. |
|---|---|---|---|---|---|
| 41 | Ptet* | B0032 | StayGold GFP | DT5 | BBa_K4491134 |
| 42 | Ptet* | B0033 | StayGold GFP | DT5 | BBa_K4491135 |
| 43 | Ptet* | B0034 | StayGold GFP | DT5 | BBa_K4491136 |
| 45 | Ptet* | B0032 | afraGFP | DT5 | BBa_K4491139 |
| 46 | Ptet* | B0032 | efasGFP | DT5 | BBa_K4491142 |
For the characterisation of the autoregulatory negative feedback circuit in the backbone pJUMP27-1A, the following Level 1 part is made
| Number | Promoter | RBS | CDS | Terminator | Registry no. |
|---|---|---|---|---|---|
| 39 | P_VanCC | B0032 | vanR^AM | MegaT | BBa_K4491132 |
For the characterisation of P_BAD promoters for part improvement in the backbone pJUMP27-1A, the following Level 1 parts is made
| Identifier | Promoter | RBS | CDS | Terminator | Registry no. |
|---|---|---|---|---|---|
| PB1 | P_BAD_AP1 | B0032 | mVenus | DT5 | BBa_K4491007 |
| PB2 | P_BAD_AP2 | B0032 | mVenus | DT5 | BBa_K4491007 |
| PB3 | P_BAD_AP3 | B0032 | mVenus | DT5 | BBa_K4491007 |
| PB4 | P_BAD_AP4 | B0032 | mVenus | DT5 | BBa_K4491007 |
| PB5 | P_BAD_AP5 | B0032 | mVenus | DT5 | BBa_K4491007 |
| PB6 | P_BAD_AP6 | B0032 | mVenus | DT5 | BBa_K4491007 |
| PB7 | P_BAD_AP7 | B0032 | mVenus | DT5 | BBa_K4491007 |
| PB8 | P_BAD_AP8 | B0032 | mVenus | DT5 | BBa_K4491007 |
| PB9 | P_BAD_AP9 | B0032 | mVenus | DT5 | BBa_K4491007 |
| PB10 | BBa_K2442101 | B0032 | mVenus | DT5 | BBa_K4491007 |
Level 1 parts are incorporated into pJUMP47-2A to form the antithetic integral controller circuit. The following table shows the circuits that we have planned to build. However, due to the interest of time, we were unable to successfully build any of them.
| Number | Function | Module A (Level 1 Number) | Module B (Level 1 Number) | Module C (Level 1 Number) | Module D (Level 1 Number) |
|---|---|---|---|---|---|
| 1 | Antithetic integral controller (ECF20, placeholder) | 1 | 3 | 8 | 24 |
| 2 | Antithetic integral controller (ECF20, placeholder) | 1 | 4 | 8 | 24 |
| 3 | Antithetic integral controller (ECF20, placeholder) | 1 | 5 | 9 | 24 |
| 4 | Antithetic integral controller (ECF20, placeholder) | 1 | 6 | 9 | 24 |
| 5 | Antithetic integral controller (ECF20, placeholder) | 1 | 7 | 9 | 24 |
| 6 | Antithetic integral controller (ECF20, placeholder) | 2 | 3 | 8 | 24 |
| 7 | Antithetic integral controller (ECF20, placeholder) | 2 | 4 | 8 | 24 |
| 8 | Antithetic integral controller (ECF20, placeholder) | 2 | 5 | 9 | 24 |
| 9 | Antithetic integral controller (ECF20, placeholder) | 2 | 6 | 9 | 24 |
| 10 | Antithetic integral controller (ECF20, placeholder) | 2 | 7 | 9 | 24 |
| 11 | Antithetic integral controller (ECF20, perturbation) | 1 | 3 | 8 | 25 |
| 12 | Antithetic integral controller (ECF20, perturbation) | 1 | 5 | 9 | 25 |
| 13 | Antithetic integral controller (sinR, placeholder) | 13 | 15 | 19 | 24 |
| 14 | Antithetic integral controller (sinR, placeholder) | 13 | 16 | 19 | 24 |
| 15 | Antithetic integral controller (sinR, placeholder) | 13 | 17 | 20 | 24 |
| 16 | Antithetic integral controller (sinR, placeholder) | 13 | 17 | 20 | 24 |
| 17 | Antithetic integral controller (sinR, placeholder) | 13 | 18 | 20 | 24 |
| 18 | Antithetic integral controller (sinR, placeholder) | 14 | 15 | 19 | 24 |
| 19 | Antithetic integral controller (sinR, placeholder) | 14 | 16 | 19 | 24 |
| 20 | Antithetic integral controller (sinR, placeholder) | 14 | 17 | 20 | 24 |
| 21 | Antithetic integral controller (sinR, placeholder) | 14 | 18 | 20 | 24 |
| 22 | Antithetic integral controller (sinR, perturbation) | 13 | 15 | 20 | 25 |
| 23 | Antithetic integral controller (sinR, perturbation) | 13 | 17 | 20 | 25 |
| 23 | Antithetic integral controller (ECF16, placeholder) | 27 | 29 | 34 | 24 |
| 24 | Antithetic integral controller (ECF16, placeholder) | 27 | 30 | 34 | 24 |
| 25 | Antithetic integral controller (ECF16, placeholder) | 27 | 31 | 35 | 24 |
| 26 | Antithetic integral controller (ECF16, placeholder) | 27 | 32 | 35 | 24 |
| 27 | Antithetic integral controller (ECF16, placeholder) | 27 | 33 | 35 | 24 |
| 28 | Antithetic integral controller (ECF16, placeholder) | 28 | 29 | 34 | 24 |
| 29 | Antithetic integral controller (ECF16, placeholder) | 28 | 30 | 34 | 24 |
| 30 | Antithetic integral controller (ECF16, placeholder) | 28 | 31 | 35 | 24 |
| 31 | Antithetic integral controller (ECF16, placeholder) | 28 | 31 | 35 | 24 |
| 32 | Antithetic integral controller (ECF16, placeholder) | 28 | 32 | 35 | 24 |
| 33 | Antithetic integral controller (ECF16, placeholder) | 28 | 33 | 35 | 24 |
| 34 | Antithetic integral controller (ECF16, perturbation) | 27 | 33 | 35 | 25 |
| 35 | Antithetic integral controller (ECF16, perturbation) | 27 | 33 | 34 | 25 |
| 36 | Open loop (ECF20) | 1 | 11 | 48 | 49 |
| 37 | Open loop (sinR) | 13 | 22 | 50 | 49 |
| 38 | Open loop (ECF16) | 27 | 37 | 51 | 49 |
| 39 | Characterisation (ECF20) | 10 | 11 | 12 | 24 |
| 40 | Characterisation (sinR) | 21 | 22 | 23 | 25 |
| 41 | Characterisation (ECF16) | 36 | 37 | 38 | 25 |