Team:Thessaly/Parts
Parts Overview
Overview
The iGEM parts are the legacy of every Team for the future iGEM Teams. Using the
iGEM registry is the best way to build your experimental design. As Abraham Lincoln
said: “The most reliable way to predict future is to create it”. So, we must submit our
parts and fully document them for future use, to make sure that they are utilized in
the most beneficial way.
Biobricks TM are fundamental parts in iGEM projects , because they constitute the
building blocks of a subsequent genetic system.
This year our goal was to create a modular system to detect the lack of Short-Chain
Fatty Acids (SCFAs) in the gastrointestinal track. While we constructed our BioBricks ,
we used the IDT synthesis offer to integrate quickly and efficiently all the
required parts. We used some pre-existing parts and we generated some new, in order
to conduct the necessary experiments Moreover, we amplified by PCR some parts
from the iGEM kit 2019 to make changes on their sequences. Below there is a list of
all parts.
Our E.coli friendly Plasmid Collection for Golden Braid Cloning
All of the parts contain various genetic elements, like promoter, RBS, CDS , terminator. We used some common parts such as a constitutive promoter and made the desirable overhangs , but we generated parts such as FFAR2-AVRP2tail-TEV cleavable site and Tetracyclin repressor (TetR) , which are some of our favorites.
Why our collection is the best?
If you are a future iGEM team this collection of plasmids gives you the
potential of full cloning ability having only 2 restriction enzymes , BsmBI and
BsaI.
GoldenBraid (GB) is a DNA assembly strategy for Plant Synthetic Biology
based on Type IIS enzymes. It is also compatible for MoClo assembly.
The sequences must not contain BsmBI and BsaI sites! Domestication may be
done in order to vanish BsaI and BsmBI sites from the inner sequence.
GB proposes an alternative view of modular cloning, and essentially the change is that you can infinitely assemble new vectors by performing “braids”(Sarrion-Perdigones et al., 2013). Using BsmBI another big advantage is the use of a single level 0 vector (pUPD and pUPD2, where pUPD2 is derived from iGEM-borne pSB1C3) for any GB part one needs.
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 ‘Le ting vel alpha’ (Level a)
But this is not the end, GoldenBraid outperforms Golden Gate (which
everyone knows) because of the ability to continuously clone TUs in an
“exponential” manner, compared to the linear progression of Golden Gate.
Binary assembly of 2 Level Ω (Level 2 for MoClo) result in an alpha vector.
Again Binary assembly of 2 alpha result 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.
What we did?
We constructed level α and level Ω plasmids with LacZa insert for blue-white screening. E.coli friendly SEVA plasmids were used as backbones and LacZa insert was taken from the original GB 2.0 pDGB1 vectors . Resulting in vectors with the pDGB1 cassete and restriction enzymes (BsaI, BsmBI). pDGB1 vectors have features for plants. On the other hand SEVA plasmids are applicable in all procaryotes (Martínez-García et al., 2019). SEVA plasmids are segmented in parts with rare restriction enzymes such us AscI, FseI, PashAI, PacI bordering the features of the plasmid. Two restriction enzymes are between the antibiotic, oriT, replication,etc. Each plasmid can be deconstructed and reconstructed with desirable parts. Also SEVA plasmids are readable in SBOL (Synthetic Biology Open Language).
Combining the best cloning assembly with the most modular backbones available, we made THE BEST PART COLLECTION of plasmids.
We also submitted to the iGEM registry the Universal Domesticator, pUPD2, for Level 0 cloning.
| BioBrick | Type | Description | Ori | Antibiotic | Length |
| BBa_K3505007 | pUPD2 | Level 0 | pMB1 | Chloramphenicol | 2690 |
| BBa_K3505009 | Alpha 2 | Level α | pBBR1 | Kanamycin | 3469 |
| BBa_K3505008 | Alpha 1R | Level α | pBBR1 | Kanamycin | 3471 |
| BBa_K3505010 | Omega 1R | Level Ω | pBBR1 | Spectinomycin | 3774 |
| BBa_K3505011 | Omega 2 | Level Ω | pBBR1 | Spectinomycin | 3521 |
NEW BASIC PARTS
All basic parts were domesticated for the GoldenBraid assembly method. Proper 5’ and 3’ overhangs based on the GoldenBraid grammar were ensured in order to facilitate type IIS restriction enzyme-mediated assembly, and thus give rise to structurally functionally correct transcription units and modules.
| BioBrick | Name | Type | Description | Length (bp) |
| BBa_K3505012 | AndersonJ23115:RBS GB compatible with A1-B3 | promoter | Constitutive promoter with RBS | 55 |
| BBa_K3505013 | AndersonJ23115:LacO:RBS A1-B3 | promoter | Constitutive promoter with lac operator and RBS | 72 |
| BBa_K3505014 | AndersonJ23115:TetO:RBS GB compatible with A1-B3 | promoter | Constitutive promoter with tet operator and RBS | 74 |
| BBa_K3505015 | prpBCDE:RBS GB compatible with A1-B3 | promoter | Promoter induced by fatty acids and more specifically by propionate | 196 |
| BBa_K3505016 | araC-ParaBAD:RBS GB compatible withA1-B2 | promoter | Arabinose inducible | 1243 |
| BBa_K3505017 | Double Terminator GB compatible with B6-C1 | terminator | Double terminator consists of rrnB T1 terminator and T7Te terminator | 137 |
| BBa_K3505018 | sfGFP GB compatible with B3-B5 | reporter | Super folded GFP , fully optimized for high quality colorimetric | 722 |
| BBa_K3505019 | eGFP GB compatible with B3-B5 | reporter | Enhanced GFP for colorimetric measurements | 725 |
| BBa_K3505020 | ECFP GB compatible with B3-B5 | reporter | Enhanced CFP for colorimetric measurements | 725 |
| BBa_K3505006 | Tyr1-AIDAc GB compatible with B3-B5 | reporter | Tyrosinase 1 fused on an, E. Coli native, membrane protein. Its role is to convert L-Tyrosine to L-DOPA. | 2546 |
| BBa_K3505005 | TetR- Tet repressor GB compatible with B3-B5 | CDS | Binds to Tet operator and inhibits transcription of the following CDS | 626 |
| BBa_K3505003 | LacI- Lac repressor GB compatible with B3-B5 | CDS | Binds to lac operator and inhibits transcription of the following CDS | 1088 |
| BBa_K3505021 | DjlA- DnaJ-like protein A GB compatible with B2-B5 | CDS | A DnaK cochaperone for resistance in membrane-induced toxicity. | 822 |
| BBa_K3505022 | RraA- Regulator of ribonuclease activity A GB compatible with B2-B5 | CDS | Prevents the degradation of the mRNA mediated RNAase E in E.coli | 492 |
| BBa_K3505023 | FFAR2:V2tail:TCS GB compatible with B2-B3 | CDS | FFAR2 GPCR that is activated from all three SCFAs , fused to a V2tail for recruitment of b-arrestin. TCS- TEV cleavage site | 1103 |
| BBa_K3505024 | B-arrestin:TEV Protease GB compatible with B2-B5 | CDS | B-arrestin fused to TEV protease that then cleaves and releases a transcriptional factor | 1959 |
New composite
| Name | Part type | Description | Composition | Length (bp) |
| BBa_K3505025 | Composite Level a assembly | Arabinose-induced Free fatty acid receptor subtype 2 chimera attached with a lac repressor protein via a TEV protease cleavable site | ParaBAD:RBS-FFAR2:AVPR2 tail:TCS-LacI-terminator | 3571 |
| BBa_K3505026 | Composite Level a assembly | Arabinose-induced TEV tagged β-arrestin-2 expression | ParaBAD:RBS- β-arrestin2:TEVp:terminator | 3339 |
| BBa_K3505027 | Composite Level a assembly | SCFAs-induced promoter FliC placed upstream of the lacI transcriptional repressor | PfliC:RBS-lac repressor:terminator | 1310 |
| BBa_K3505028 | Composite Level a assembly | Cyan-based reporter system for the purpose of characterizing and measuring the activity of the PfliC promoter | PfliC:RBS-eCFP-terminator | 947 |
| BBa_K3505029 | Composite Level a assembly | Reporter system utilizing the enhanced fluorescent protein, for the purpose of characterizing and measuring the activity of the PfliC promoter | PfliC:RBS - eGFP-terminator | 947 |
| BBa_K3505030 | Composite Level a assembly | Reporter system based on the super-folder green fluorescent protein for the purpose of characterizing and measuring the activity of the PfliC promoter | PfliC:RBS- sfGFP-terminator | 944 |
| BBa_K3505031 | Composite Level a assembly | Propionate-induced prpBCDE promoter placed upstream of a lac repressor | prpBCDE:RBS-lac repressor-terminator | 1421 |
| BBa_K3505032 | Composite Level a assembly | Downstream fluorescence reporter system regulated by the transcription repressor lacI | pAndersonJ23115:lacO:RBS-eCFP -terminator | 934 |
| BBa_K3505033 | Composite Level a assembly | Constitutively expressed of the tetracycline repressor protein | pAndersonJ23115:RBS:tetR-terminator | 827 |
| BBa_K3505034 | Composite Level a assembly | tetracycline-controlled regulation of the expression of the lacI, via the regulation of tetR | pAndersonJ23115:tetO:RBS-lacI-terminator | 1297 |
| BBa_K3505035 | Composite Level a assembly | Constitutively expressed membrane embedded tyrosinase serving as an electrochemical reporter system | pAndersonJ23115:RBS-tyrosinase:AIDA-terminator-terminator | 2754 |
| BBa_K3505038 | Composite Level a assembly | Arabinose inducible anti-mRNA degrading protein | ParaBAD-RraA-Terminator | 1872 |
| BBa_K3505039 | Composite Level a assembly | Arabinose inducible cochaperon for better folding of proteins | ParaBAD-DjlA-Terminator | 2218 |
| BBa_K3505036 | Composite Level Ω assembly | pSEVA Ω1R vector incorporating the Prom Assay | pFliC:RBS-LacI-Terminator- pAndersonJ23115:LacO:RBS-ECFP-terminator | 2244 |
| BBa_K3505037 | Composite Level Ω assembly | pSEVA Ω1R vector incorporating the tet-OFF system | pAndersonJ23115:RBS-TetR-terminator- pAndersonJ23115:tetO:RBS-LacI-terminator | 2124 |
References
Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez (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
Esteban Martínez-García, Angel Goñi-Moreno, Bryan Bartley, James McLaughlin, Lucas Sánchez-Sampedro, Héctor Pascual del Pozo, Clara Prieto Hernández, Ada Serena Marletta, Davide De Lucrezia, Guzmán Sánchez-Fernández, Sofía Fraile, Víctor de Lorenzo, SEVA 3.0: an update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts, Nucleic Acids Research , Volume 48, Issue D1, 08 January 2020, Pages D1164–D1170, https://doi.org/10.1093/nar/gkz1024
