Team:Thessaly/Part Collection
Parts Collection
Our E.coli friendly Plasmid Collection for GoldenBraid 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 as 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 |
After the apply of the preliminary Judging Form for the Best Part Collection we had a
problem that our plasmids had the type IIS sites in their sequence because they play
crucial role for th GoldenBraid Assembly. So we submitted
separately the backbones and the inserts.
We also Submitted a variety of very common of Level 0 interchangeable parts.
Figure 2. The LacZa modules with the proper BsmBI and BsaI sites
| BioBrick | Type | Description | Ori | Antibiotic | Length |
| BBa_K3 505043 | pUPD2 BACKBONE | Level 0 | pMB1 | Chloramphenicol | 2093 |
| BBa_K3 505040 | Alpha BACKBONE | Level α | pBBR1 | Kanamycin | 2841 |
| BBa_K3 505042 | Omega BACKBONE | Level Ω | pBBR1 | Spectinomycin | 2894 |
| BBa_K3 505041 | LacZa | Insert | - | - | 627 |
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
