One of the main goals of our team was to create a collection of parts that could facilitate future teams to make their ideas come true. Therefore, we constructed a library of 57 parts in total, from which 51 were designed by our team, 5 were provided by external laboratories and 1 was already existing in the Registry (BBa_K3505007).
Our Basic Parts come with proposed 4nt-overhangs, compatible with the GoldenBraid 2.0 Cloning System, in order to be easy for other teams to use them and combine them as they like.
1st Module
The 1st module of our design is supposed to contain a detection system (microcystin-LR riboswitch) and a repressor system (tetr gene). To characterize this module to a good extent and to create the parts needed for our Composite Parts, we designed a great number of Basic Parts, presented below:
| Name | Description | Golden Braid Overhangs | Designer | Length (bp) |
|---|---|---|---|---|
| BBa_K4213022 | NOS promoter (for TetR) | A1 – B2 | Thodoris Papanikolas, Eleftheria Lakaki | 331 |
| BBa_K4213023 | NOS promoter (for Venus-TetR) | A1 – B1 | Thodoris Papanikolas | 325 |
| BBa_K4213012 | mVenus Q69M (for TetR) | B2 | Eleftheria Lakaki, Thodoris Papanikolas | 757 |
| BBa_K4213000 | TPP Riboswitch | B6 – C1 | Yannos Retalis | 710 |
| BBa_K4213018 | TetR optimized – NLS (for Venus) | B3 – B5 | Thodoris Papanikolas | 683 |
| BBa_K4213019 | TetR optimized – NLS – KRAB (for Venus) | B3 – B4 | Thodoris Papanikolas | 679 |
| BBa_K4213001 | TetR optimized – NLS | B3 – B5 | Thodoris Papanikolas, Eleftheria Lakaki | 686 |
| BBa_K4213028 | TetR optimized – NLS - KRAB | B3 - B4 | Thodoris Papanikolas, Eleftheria Lakaki | 682 |
| BBa_K4213027 | NOS terminator | B6 – C1 | Provided by the Laboratory of Plant & Environmental Biotechnology (Dep. Of Biochemistry & Biotechnology, University of Thessaly) | 500 |
2nd Module
The 2nd module is a transcriptional unit that is regulated by the 1st module and from which a PHT1 protein is expressed. For the characterization of this protein and the later construction of Composite Parts, the following Basic Parts were designed:
| Name | Description | Golden Braid Overhangs | Designer | Length (bp) |
|---|---|---|---|---|
| BBa_K4213026 | TetO7 (7 repeats of TetO element) | A1 | Thodoris Papanikolas, Yannos Retalis | 426 |
| BBa_K4213003 | 35S promoter – TripleTetO | A2 – B2 | Thodoris Papanikolas | 1127 |
| BBa_K4213002 | PHT1;5 from A. thaliana optimized - linker | B3 - B4 | Eleftheria Lakaki, Thodoris Papanikolas | 1672 |
| BBa_K4213004 | PHT1;5 from A. thaliana non optimized - linker | B3 – B4 | Eleftheria Lakaki, Thodoris Papanikolas | 1672 |
| BBa_K4213021 | PHT1;6 from O. sativa optimized + domesticated - linker | B3 – B4 | Eleftheria Lakaki, Thodoris Papanikolas | 1648 |
| BBa_K4213007 | PHT1;6 from O. sativa partial optimized - linker | B3 – B4 | Eleftheria Lakaki, Thodoris Papanikolas | 1651 |
| BBa_K4213010 | mVenus Q69M (for PHT1) | B5 | Eleftheria Lakaki | 758 |
| BBa_K4213025 | NOS terminator - stuffer | B6 - C1 | Eleftheria Lakaki | 692 |
Common Fragments
The parts, listed below, were used for the construction of both modules.
| Name | Description | Golden Braid Overhangs | Designer | Length (bp) |
|---|---|---|---|---|
| BBa_K4213011 | mVenus Q69M (cds) | B3 – B5 | Eleftheria Lakaki | 755 |
| BBa_K4213024 | HSP terminator – NOS terminator | B6 – C1 | Eleftheria Lakaki | 541 |
The design of our parts was based on the GoldenBraid Cloning System to ensure an increased usability thanks to the double iterative loop of this assembly system.
That means that, our level α parts consist of the Basic Parts combined to form transcriptional units. Two level α transcriptional units can be assembled together leading to the formation of two alternative level Ω constructs, which could also form later level α parts (not in our case).
Level Alpha (α)
| Name | Module | Description | Designer | Length (bp) |
|---|---|---|---|---|
| BBa_K4213029 | 1st | NOS promoter - mVenus Q69M - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 1680 |
| BBa_K4213030 | 1st | NOS promoter - mVenus Q69M- TetR optimized – NLS - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 2325 |
| BBa_K4213031 | 1st | NOS promoter - mVenus Q69M - HSP terminator - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 1529 |
| BBa_K4213032 | 1st | NOS promoter - mVenus Q69M- TetR optimized – NLS - HSP terminator - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 2174 |
| BBa_K4213033 | 1st | NOS promoter - mVenus Q69M- TetR optimized – NLS - KRAB - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 2704 |
| BBa_K4213034 | 1st | NOS promoter - mVenus Q69M- TetR optimized – NLS - KRAB - HSP terminator - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 2553 |
| BBa_K4213044 | 1st | NOS promoter - TetR optimized – NLS - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 1453 |
| BBa_K4213045 | 1st | NOS promoter - TetR optimized – NLS - KRAB - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 1831 |
| BBa_K4213046 | 1st | NOS promoter - TetR optimized – NLS - HSP terminator - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 1302 |
| BBa_K4213047 | 1st | NOS promoter - TetR optimized – NLS - KRAB - HSP terminator - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 1680 |
| BBa_K4213035 | 2nd | TetO7 - 35S promoter - PHT1;5 from A. thaliana optimized - linker - mVenus Q69M- NOS terminator - stuffer | Eleftheria Lakaki, Thodoris Papanikolas | 4509 |
| BBa_K4213036 | 2nd | TetO7 - 35S promoter - PHT1;5 from A. thaliana non optimized - linker - mVenus Q69M- NOS terminator - stuffer | Eleftheria Lakaki, Thodoris Papanikolas | 4509 |
| BBa_K4213037 | 2nd | TetO7 - 35S promoter - PHT1;5 from A. thaliana non optimized - linker - mVenus Q69M - HSP terminator - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 4358 |
| BBa_K4213038 | 2nd | TetO7 - 35S promoter - PHT1;6 from O. sativa optimized + domesticated - linker - mVenus Q69M- HSP terminator - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 4334 |
| BBa_K4213039 | 2nd | TetO7 - 35S promoter - PHT1;6 from O. sativa partial optimized - linker - mVenus Q69M- HSP terminator - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 4337 |
| BBa_K4213040 | 2nd | TetO7 - 35S promoter - PHT1;6 from O. sativa optimized + domesticated - linker - mVenus Q69M- NOS terminator - stuffer | Eleftheria Lakaki, Thodoris Papanikolas | 4485 |
| BBa_K4213041 | 2nd | TetO7 - 35S promoter - mVenus Q69M- NOS terminator - stuffer | Eleftheria Lakaki, Thodoris Papanikolas | 2868 |
| BBa_K4213042 | 2nd | TetO7 - 35S promoter - PHT1;5 from A. thaliana optimized - linker - mVenus Q69M - HSP terminator - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 4358 |
| BBa_K4213043 | 2nd | TetO7 - 35S promoter - PHT1;6 from O. sativa partial optimized - linker - mVenus Q69M - NOS terminator - stuffer | Eleftheria Lakaki, Thodoris Papanikolas | 4488 |
Level Omega (Ω)
We constructed several level Ω parts for different purposes. Firstly, we designed parts for experiments involving the Improvement of the part BBa_C0040 and, secondly, for the test and further characterization of our whole system.
| Name | Purpose | Description | Designer | Length (bp) |
|---|---|---|---|---|
| BBa_K4213048 | Improvement of an Existing Part | TetO7 - 35S promoter - mVenus Q69M - NOS terminator - stuffer - NOS promoter - TetR - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 4208 |
| BBa_K4213049 | Improvement of an Existing Part | TetO7 - 35S promoter - mVenus Q69M - NOS terminator - stuffer - NOS promoter - TetR - KRAB - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 4587 |
| BBa_K4213050 | Characterization of our system | TetO7 - 35S promoter - PHT1;5 from A. thaliana optimized - linker - mVenus Q69M - NOS terminator - stuffer - NOS promoter - TetR - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 5849 |
| BBa_K4213051 | Characterization of our system | TetO7 - 35S promoter - PHT1;5 from A. thaliana non optimized - linker - mVenus Q69M - NOS terminator - stuffer - NOS promoter - TetR - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 5849 |
| BBa_K4213052 | Characterization of our system | TetO7 - 35S promoter - PHT1;6 from O. sativa optimized + domesticated - linker - mVenus Q69M - NOS terminator - stuffer - NOS promoter - TetR - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 5825 |
| BBa_K4213053 | Characterization of our system | TetO7 - 35S promoter - PHT1;6 from O. sativa partial optimized - linker - mVenus Q69M - NOS terminator - stuffer - NOS promoter - TetR - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 5825 |
| BBa_K4213054 | Characterization of our system | TetO7 - 35S promoter - PHT1;5 from A. thaliana optimized - linker - mVenus Q69M - NOS terminator - stuffer - NOS promoter - TetR - KRAB - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 6228 |
| BBa_K4213055 | Characterization of our system | TetO7 - 35S promoter - PHT1;5 from A. thaliana non optimized - linker - mVenus Q69M - NOS terminator - stuffer - NOS promoter – TetR - KRAB - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 6228 |
| BBa_K4213056 | Characterization of our system | TetO7 - 35S promoter - PHT1;6 from O. sativa optimized + domesticated - linker - mVenus Q69M - NOS terminator - stuffer - NOS promoter - TetR - KRAB - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 6204 |
| BBa_K4213057 | Characterization of our system | TetO7 - 35S promoter - PHT1;6 from O. sativa partial optimized - linker - mVenus Q69M - NOS terminator - stuffer - NOS promoter - TetR - KRAB - NOS terminator | Eleftheria Lakaki, Thodoris Papanikolas | 6204 |
The destination vectors for our constructs are universal domesticator plasmids that are usually used in synthetic biology and for domestication of DNA parts for GoldenBraid assembly.
| Name | Description | Origin | Length (bp) |
|---|---|---|---|
| BBa_K3505007 | pUPD2 | iGEM Thessaly 2020 | 2690 |
| BBa_K4213058 | pDGB3alpha1 | Provided by the Laboratory of Plant & Environmental Biotechnology (Dep. Of Biochemistry & Biotechnology, University of Thessaly) | 6965 |
| BBa_K4213059 | pDGB3alpha2 | Provided by the Laboratory of Plant & Environmental Biotechnology (Dep. Of Biochemistry & Biotechnology, University of Thessaly) | 6967 |
| BBa_K4213060 | pDGB3omega1 | Provided by the Laboratory of Plant & Environmental Biotechnology (Dep. Of Biochemistry & Biotechnology, University of Thessaly) | 7295 |
| Name | Description | Designer | Length (bp) |
|---|---|---|---|
| BBa_K4213008 | Forward Primer for TetO7 | Yannos Retalis | 36 |
| BBa_K4213009 | Reverse Primer for TetO7 | Yannos Retalis | 36 |
| BBa_K4213014 | Forward primer for domestication of aHHR-OCS3 sequence (Set 1) | Yannos Retalis | 46 |
| BBa_K4213015 | Reverse primer for domestication of aHHR-OCS3 sequence (Set 2) | Yannos Retalis | 39 |
| BBa_K4213016 | Forward primer for domestication of aHHR-OCS3 sequence (Set 2) | Yannos Retalis | 33 |
| BBa_K4213017 | Reverse primer for domestication of aHHR-OCS3 sequence (Set 1) | Yannos Retalis | 36 |
| Name | Type | Description | Length (bp) |
|---|---|---|---|
| BBa_K4213013 | Plasmid | pENTR-GFP-aHHR_theophylline | 4357 |
We hope that our contribution to the enrichment of the Registry will be useful for future iGEM teams.