Team:Thessaly/Parts
Overview
Without libraries what do we have? We have no past and no future. - Ray Bradbury
As an iGEM team we have the duty to leave a mark and tools for future iGEM teams. We aimed to achieve a functional project based on modular systems composed of interchangeable parts. We used 17 existing parts and constructed the following 15 basic and 20 composite parts. Two parts of the registry were characterized, Promoter FliC BBa_K2924016 and BBa_K415202 CymR. Also, we improved bglX (BBa_K523002) and propose N20bglX GB compatible with B3-B5 (BBa_K3866032).
As an iGEM team we have the duty to leave a mark and tools for future iGEM teams. We aimed to achieve a functional project based on modular systems composed of interchangeable parts. We used 17 existing parts and constructed the following 15 basic and 20 composite parts. Two parts of the registry were characterized, Promoter FliC BBa_K2924016 and BBa_K415202 CymR. Also, we improved bglX (BBa_K523002) and propose N20bglX GB compatible with B3-B5 (BBa_K3866032).
Introduction
During our project, the team used 17 existing parts and constructed the following 15 basic
and 20 composite parts. These parts are suitable for
GoldenBraid cloning (GB). GB is a modular DNA
assembly strategy for Plant Synthetic Biology based on Type IIS enzymes, also compatible with MoClo
assembly. It proposes an alternative view of modular cloning, and essentially you can infinitely
assemble new vectors by performing “braids” (Sarrion-Perdigones et al., 2013). All the components in
the
GoldenBraid system are free of internal BsaI and BsmBI sites. Domestication may be done in order
to vanish BsaI and BsmBI sites from the inner sequence.
We use BsmbI for a single level 0 vector (pUPD2, where pUPD2 is derived from iGEM-borne pSB1C3) for any GB part one needs.
Figure 1: The
GoldenBraid Grammar
Then, combining the desirable fragments from level 0, ‘level alpha’ (level a) cloning is succeeded, creating Transcription Units (TU). Desirable TUs are combined to result in (Level Ω) cloning.
Binary assembly of 2 Level Ω (Level 2 for MoClo) results in an alpha vector. Again Binary assembly of 2 alpha results in an omega vector. With this assembly you can insert step by step as many parts and as many TUs you want with high efficiency. GoldenBraid outperforms Golden Gate because of the ability to continuously clone TUs in an “exponential” manner, compared to the linear progression of Golden Gate. The clonings were done in SEVA (Martínez-García, et al,2015) (Standard European Vector Architecture) plasmids compatible with Bacterial GB we constructed last year (Damalas et al. 2020).
We use BsmbI for a single level 0 vector (pUPD2, where pUPD2 is derived from iGEM-borne pSB1C3) for any GB part one needs.
Figure 1: The
GoldenBraid Grammar
Then, combining the desirable fragments from level 0, ‘level alpha’ (level a) cloning is succeeded, creating Transcription Units (TU). Desirable TUs are combined to result in (Level Ω) cloning.
- Using BsmBI for Level 0 modules and ‘level omega’ (Level Ω).
- Using BsaI for ‘Level alpha’ (Level a).
Binary assembly of 2 Level Ω (Level 2 for MoClo) results in an alpha vector. Again Binary assembly of 2 alpha results in an omega vector. With this assembly you can insert step by step as many parts and as many TUs you want with high efficiency. GoldenBraid outperforms Golden Gate because of the ability to continuously clone TUs in an “exponential” manner, compared to the linear progression of Golden Gate. The clonings were done in SEVA (Martínez-García, et al,2015) (Standard European Vector Architecture) plasmids compatible with Bacterial GB we constructed last year (Damalas et al. 2020).
Favorite Thessaly 2021 iGEM Team Parts
| Name | Type | Description | Designer | Length | |||
|---|---|---|---|---|---|---|---|
| W | BBa_K3866032 | Coding | N20bglΧ GB compatible with B3-B5 | Efthymia Zisopoulou | 2321 | ||
| X | BBa_K3866033 | Composite | pAndersonJ23115:RBS-bglX-terminator | Efthymia Zisopoulou | 2546 | ||
| W | BBa_K3866037 | Composite | pAndersonJ23115:RBS-N20bglx-terminator | Efthymia Zisopoulou | 2529 |
Thessaly 2021 iGEM Team Parts Sandbox
| Name | Type | Description | Designer | Length | |||
|---|---|---|---|---|---|---|---|
| BBa_K3866000 | Regulatory | araC-ParaBAD:RBS GB compatible with A1-B2 | Venetios Michelioudakis Anna Patri | 1243 | |||
| BBa_K3866001 | Regulatory | pTrc:RBS GB compatible with A1-B2 | Pericles Vasileiou | 74 | |||
| BBa_K3866002 | Coding | ackA-Acetate kinase GB compatible with B3-B5 | Venetios Michelioudakis | 1211 | |||
| BBa_K3866003 | Regulatory | Partial T5 Promoter - CuO- RBS GB compatible with A1-B2 | Ioanna Gkoni | 97 | |||
| BBa_K3866004 | Coding | pta- Phosphate acetyltransferase GB compatible with B3-B5 | Venetios Michelioudakis | 2150 | |||
| BBa_K3866005 | Regulatory | Plac-RBS GB compatible with A1-B2 | Ioanna Gkoni | 49 | |||
| BBa_K3866006 | Translational_Unit | ParaBAD-ackA-Terminator | Venetios Michelioudakis | 2607 | |||
| BBa_K3866007 | Translational_Unit | ParaBAD-pta-Terminator | Venetios Michelioudakis | 3546 | |||
| BBa_K3866008 | Coding | MazE antitoxin GB compatible with B3-B5 | Ioanna Gkoni | 254 | |||
| BBa_K3866009 | Composite | Acetate production construct | Venetios Michelioudakis | 4120 | |||
| BBa_K3866010 | Translational_Unit | ParaBAD-tyrosinase:AIDA-terminator | Anna Patri | 3104 | |||
| BBa_K3866011 | Coding | MazF toxin GB compatible with B3-B5 | Ioanna Gkoni | 341 | |||
| BBa_K3866012 | Composite | ParaBAD:RBS-FFAR2:AVPR2 tail:TCS-LacI-terminator---ParaBAD-RraA-Terminator | Venetios Michelioudakis | 5451 | |||
| BBa_K3866013 | Composite | ParaBAD:RBS- β-arrestin2:TEVp:terminator---pAndersonJ23115:lacO:RBS-eCFP -terminator | Venetios Michelioudakis | 4281 | |||
| BBa_K3866014 | Composite | TANGO module GPCR and B arrestin | Venetios Michelioudakis | 9740 | |||
| BBa_K3866015 | Coding | PhaA-RBS-PhaB-RBS-Crt-RBS-Ter GB compatible with B3-B5 | Ioanna Gkoni | 3968 | |||
| BBa_K3866016 | Translational_Unit | Plac-MazE-double terminator | Ioanna Gkoni | 456 | |||
| BBa_K3866017 | Translational_Unit | PArtial T5 - CuO - MazF - double terminator | Ioanna Gkoni | 591 | |||
| BBa_K3866018 | Translational_Unit | J23115-RBS-CymR-double terminator | Ioanna Gkoni | 818 | |||
| BBa_K3866019 | DNA | Stuffer PDGB A2 | Ioanna Gkoni | 162 | |||
| BBa_K3866020 | Composite | J23115-CymR-Stuffer a2 | Ioanna Gkoni | 988 | |||
| BBa_K3866021 | Composite | ParaBAD:RBS-Superpart 1-double terminator | Ioanna Gkoni | 5364 | |||
| BBa_K3866022 | Regulatory | pLac:RBS GB compatible with A1-B2 | Pericles Vasileiou | 75 | |||
| BBa_K3866023 | Coding | sbm - Methylmalonyl-CoA mutase GB compatible with B2-B5 | Pericles Vasileiou | 2150 | |||
| BBa_K3866024 | Coding | ygfG - Methylmalonyl-CoA decarboxylase GB compatible with B2-B5 | Pericles Vasileiou | 791 | |||
| BBa_K3866025 | Coding | ygfH - Propionyl-CoA:succinate CoA transferase GB compatible with B2-B5 | Pericles Vasileiou | 1484 | |||
| BBa_K3866026 | Translational_Unit | ParaBAD:sbm:Terminator | Pericles Vasileiou | 3546 | |||
| BBa_K3866027 | Translational_Unit | ParaBAD:ygfG:Terminator | Pericles Vasileiou | 2187 | |||
| BBa_K3866028 | Translational_Unit | ParaBAD:ygfH:Terminator | Pericles Vasileiou | 2880 | |||
| BBa_K3866029 | Composite | ParaBAD:sbm:Terminator - stuffer | Pericles Vasileiou | 3716 | |||
| BBa_K3866030 | Composite | ParaBAD:ygfG:Terminator - ParaBAD:ygfH:Terminator | Pericles Vasileiou | 5075 | |||
| BBa_K3866031 | Composite | Propionate Production Construct | Pericles Vasileiou | 8799 |
EXISTING PARTS USED
| Biobrick | Description | Team |
| pFlic for SCFAs detection | ||
| BBa_K3505030 | PfliC:RBS- sfGFP-terminator | Thessaly 2020 |
| BBa_K3505028 | PfliC:RBS-eCFP-terminator | Thessaly 2020 |
| BBa_K3505036 | NOT GATE PfliC | Thessaly 2020 |
| Tyrosinase for readout | ||
| BBa_K3505006 | Tyr1-AIDAc | Thessaly 2020 |
| BBa_K3505017 | Double Terminator | Thessaly 2020 |
| BglX for increased carbon flux | ||
| BBa_K523002 | periplasmic BglX | Edinburgh 2011 |
| BBa_K3505012 | pAnderson J23115:RBS | Thessaly 2020 |
| Cymr for Probiotic Kill Switch | ||
| BBa_K415202 | CymR | MIT 2012 |
| GPCR for SCFAs detection | ||
| BBa_K3505025 | ParaBAD:RBS-FFAR2:AVPR2 tail:TCS-LacI-terminator | Thessaly 2020 |
| BBa_K3505026 | ParaBAD:RBS- β-arrestin2:TEVp:terminator | Thessaly 2020 |
| BBa_K3505038 | ParaBAD-RraA-Terminator | Thessaly 2020 |
| BBa_K3505032 | pAndersonJ23115:lacO:RBS-eCFP -terminator | Thessaly 2020 |
| Plasmids for cloning and expression | ||
| BBa_K3505007 | pUPD2 | Thessaly 2020 |
| BBa_K3505008 | seva GB alpha 1R (α1R) | Thessaly 2020 |
| BBa_K3505009 | seva GB alpha 2 (α2) | Thessaly 2020 |
| BBa_K3505010 | seva GB omega 1R (Ω1R) | Thessaly 2020 |
| BBa_K3505011 | seva GB omega 2 (Ω2) | Thessaly 2020 |
CONTRIBUTION
Two parts of the registry were characterized by adding new documentation from bibliography and/or our
experiments. Trying to strengthen the project design we chose to characterize Promoter FliC
BBa_K2924016
which takes place in the SCFAs detection module, adding more documentation and testing
more conditions to a part that was used by our team last year, in Amalthea 2020. We also made a
literature documentation on BBa_K415202 CymR, which is the center of our probiotic Kill switch.
IMPROVEMENT
The probiotic supplement that we propose has the ability to degrade cellulose. In order to achieve
that, we decided to improve one of the three enzymes that are responsible for degradation of
cellobiose, the repeating unit of cellulose. The improved part is N20bglΧ GB compatible with
B3-B5 (BBa_K3866032) based upon the bglX (BBa_K523002).
The parts that our wet lab team used,
for the improvement experiments, are the AndersonJ23115:RBS-bglX-terminator (BBa_K3866033) and
pAndersonJ23115:RBS-N20bglx-terminator (BBa_K3866037).
References
- Damalas, S., Batianis, C., Martin‐Pascual, M., Lorenzo, V., & Martins dos Santos, V. (2020). SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards. Microbial Biotechnology, 13(6), 1793-1806. doi: 10.1111/1751-7915.13609
- Martínez-García, E., Aparicio, T., Goñi-Moreno, A., Fraile, S., & de Lorenzo, V. (2015). SEVA 2.0: an update of the Standard European Vector Architecture for de-/re-construction of bacterial functionalities. Nucleic acids research, 43(Database issue), D1183–D1189. https://doi.org/10.1093/nar/gku1114
- Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., & Ziarsolo, P. et al. (2013). GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. PLANT PHYSIOLOGY, 162(3), 1618-1631. doi: 10.1104/pp.113.217661