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Jamboree
Throughout our iGEM journey, we learned how to utilize various bioparts and integrate them like pieces of a puzzle to construct transcriptional units and a bacterial system that performs as we intended. We combined different cloning methods (hyperlink for engineering) and used various vectors to successfully express our system in different chassis, namely E. coli and P. putida. To accomplish our objectives with THAELIA, we designed 24 basic parts and 34 composite parts. Of these, we tested 14 basic parts, which were then combined to create 16 composite parts. On this page, you’ll find the parts and the constructs we tested, along with additional elements such as the primers we designed and the cloning vectors we utilized.
Our Part Collection serves as a place to have the entirety of parts used, either from the iGEM Registry or from our literature review, for them to be easily accessible. Our Part collection acts essentially as Project’s THAELIA very own Registry. We have also added our own composite parts, for documentation purposes, as well as a way of Contribution, as they consist of units ready to be expressed. This saves time for future teams in their own design and ordering phase.
For our cloning, we used a variety of vectors, since we worked with two different chassis. Firstly, for E.coli strains (DH5a, BL21 (DE3)) we used Golden Braid compatible vectors, a method traditionally used by iGEM Thessaly teams. The domestication was done according to the Golden Braid grammar, using the tool Golden Braid domesticator.
Secondly, this year our team decided to venture out to another cloning method, the Golden Standard method, a transition necessitated by our chassis, Pseudomonas putida.
You can learn more about our cloning procedure in our Engineering page:
Level 0 constructs were all created in the pUPD2 Golden Braid vector.
Level α constructs (1 transcription unit) for E.coli
were created in the pDGB3alpha1 and pDGB3alpha2 Golden Braid vectors.
Level ω constructs (2 transcription units) for E.coli
were created in the pDGB3omega1.
Level 1 constructs (1 transcription unit) for P.putida
were created in the pSEVA23g19[g1] and pSEVA23g19[g2].
Golden Braid (GB) is a powerful modular cloning system, based on Type II enzymes, enabling consistent multigene assembly with a huge range of combination possibilities [1].
Our Golden Braid Vectors:
| Name | Registry Code | Short Description |
|---|---|---|
| pUPD2 | BBa_K3505007 https://parts.igem.org/Part:BBa_K3505007 | Level 0 vector for GoldenBraid assembly, ori=MB1 |
| pDGB3alpha1 (a1) | BBa_K4213058 https://parts.igem.org/Part:BBa_K4213058 | Level α vector for GoldenBraid assembly, ori= BR322 |
| pDGB3alpha2 (a2) | BBa_K4213059 https://parts.igem.org/Part:BBa_K4213059 | Level α vector for GoldenBraid assembly, ori=BR322 |
| pDGB3omega1 (ω1) | BBa_K4213060 https://parts.igem.org/Part:BBa_K4213060 | Level ω vector for GoldenBraid assembly, ori=BR322 |
Golden Standard (GS) is a Type IIS assembly method based on the Standard European Vector Architecture (SEVA). It enables modular cloning in most proteobacteria and allows for the reliable assembly of genetic circuits with up to twenty transcription units [2].
Our Golden Standard Vectors:
| Name | Registry Code | Short Description |
|---|---|---|
| pSEVA23g19[g1] | BBa_K5299002 https://parts.igem.org/Part:BBa_K5299002 | Level 1 vector for Golden Standard assembly, ori= BBR1 |
| pSEVA23g19[g2] | BBa_K5299003 https://parts.igem.org/Part:BBa_K5299003 | Level 1 vector for Golden Standard assembly, ori= BBR1 |
| pSEVA63g19[gA] | BBa_K5299004 https://parts.igem.org/Part:BBa_K5299004 | Level 2 vector for Golden Standard assembly, ori= BBR1 |
We also attempted to integrate some of our constructs into the bacterial chromosome using the mini Tn-7 system [3]. For this, we needed the help of the following plasmids:
| Name | Registry Code | Short Description |
|---|---|---|
| pTn7-M | BBa_K5299006 https://parts.igem.org/Part:BBa_K5299006 | mini-Tn7 suicide delivery vector |
| pTNS-2 | BBa_K5299001 https://parts.igem.org/Part:BBa_K5299001 | Carries the genes encoding components of the Tn7 transposase (TnSABC+D operon |
| pRK600 | BBa_K5299005 https://parts.igem.org/Part:BBa_K5299005 | Helper plasmid |
| pTN7-19[g1] | BBa_K5299007 https://parts.igem.org/Part:BBa_K5299007 | mini-Tn7 suicide delivery vector for Golden Standard Level 1 reactions |
We utilized the Golden Braid domesticator to add 4-nucleotide overhangs into our parts. These domesticated parts were then placed in pUPD2 to assemble our level 0 parts, making it easier to combine them and create our desired constructs. All our level 0 parts were domesticated for the enzymes BsmBI, BpiI and BsaI, as we use the Type IIS Assembly method.
| Type of Part | Golden Braid overhangs |
|---|---|
| Promoter- RBS | A1 - A2 - A3 - B1 - B2 |
| CDS | B3 - B4 - B5 |
| Terminator | B6 - C1 |
In our Part Collection, we present the origin of the parts we used without the overhangs. This makes them useful for other teams to use in any type of cloning procedure by adding the required overhangs.
Promoters :
| Part Name | Registry code | Short Description |
|---|---|---|
| J23119 | BBa_J23119 https://parts.igem.org/Part:BBa_J23119 | Anderson Constitutive promoter |
| P3.1 | BBa_K4583008 https://parts.igem.org/Part:BBa_K4583008 | Auto-inducible stationary phase P3.1 promoter |
| OsmY | BBa_J45992 https://parts.igem.org/Part:BBa_J45992 | Registry catalog stationary phase promoter |
| T7 pro | BBa_I719005 https://parts.igem.org/Part:BBa_I719005 | T7 RNAP promoter long |
| T7 pro reverse | BBa_K5299020 https://parts.igem.org/Part:BBa_K5299020 | T7 RNAP promoter long reverse |
| BG37 | BBa_K5299008 https://parts.igem.org/Part:BBa_K5299008 | Auto-inducible exponential phase promoter (Zobel et al. 2015) |
| BG42 | Ba_K5299010 https://parts.igem.org/Part:BBa_K5299010 | Auto-inducible exponential phase promoter (Zobel et al. 2015) |
| BG17 | BBa_K5299009 https://parts.igem.org/Part:BBa_K5299009 | Auto-inducible exponential phase promoter (Zobel et al. 2015) |
Ribosome Binding Sites (RBSs)
| Part Name | Registry Code | Short Description |
|---|---|---|
| RBS 1 | BBa_B0030 https://parts.igem.org/Part:BBa_B0030 | strong, well characterized RBS |
| RBS 2(Elowitz 1999) | BBa_B0034 https://parts.igem.org/Part:BBa_B0034 | strong, well characterized RBS |
Coding Sequences (CDSs)
| Part name | Registry Code | Short Description |
|---|---|---|
| sfGFP | BBa_I746916 https://parts.igem.org/Part:BBa_I746916 | Superfolder green fluorescent protein |
| eTEV protease | BBa_K3989002 https://parts.igem.org/Part:BBa_K3989002 | Enhanced version of the Tobacco Etch Virus (TEV) protease |
| T7 RNAP | BBa_K1706007 https://parts.igem.org/Part:BBa_K1706007 | Highly specific and efficient enzyme that transcribes DNA from the T7 promoter |
| AAC (ATP- ADP carrier) | BBa_K5299120 https://parts.igem.org/Part:BBa_K5299120 | V.dahliae gene (NW_009276936.1:165449-167157 (Accession number- NCBI)) |
| THI20 | BBa_K5299121 https://parts.igem.org/Part:BBa_K5299121 | V.dahliae gene (XM_009655087.1 (Accession number- NCBI)) |
| RGS1 | BBa_K5299119 https://parts.igem.org/Part:BBa_K5299119 | V.dahliae gene (MG583845.1 (Accession number- NCBI)) |
Terminators
| Part name | Registry Code | Short Description |
|---|---|---|
| ter | BBa_B0015 https://parts.igem.org/Part:BBa_B0015 | double terminator |
| T7hyb6 | BBa_K5299118 https://parts.igem.org/Part:BBa_K5299118 | T7 RNAP terminator (Calvopina-Chavez et al. 2022) |
| T7 hyb6 reverse | BBa_K5299021 https://parts.igem.org/Part:BBa_K5299021 | T7 RNAP terminator reverse (Calvopina-Chavez et al. 2022) |
Our composite parts were designed following the Golden Braid 2.0 grammar. This allows us to combine our basic parts (level 0), each with compatible 3' and 5' overhangs, to form a complete transcriptional unit in a level alpha plasmid. Two level alpha plasmids, each carrying a transcriptional unit, can then be combined to create a level omega plasmid that holds both transcriptional units. These composite parts were expressed in E. coli. [1] Below, you can find all the level 1 (alpha) and level 2 (omega) composite parts we tested during this iGEM season.
Our Level a constructs (Golden Braid Assembly)
Similar to our basic parts, our composite parts were designed following the Golden Braid 2.0 assembly system, which operates using a double iterative loop. This allows level 0 basic parts, each featuring compatible 3' and 5' overhangs, to be assembled into a transcriptional unit within a level alpha plasmid. Two level alpha plasmids, each containing a distinct transcriptional unit, can then be combined to form a level omega plasmid, merging both transcriptional units. Below, we have listed all the level 1 (alpha) and level 2 (omega) composite parts that we designed and tested in silico using SnapGene, as well as those we experimentally validated in the lab during this iGEM season.
| Part name | Registry Code | GB Vector |
|---|---|---|
| P3.1 - RBS1- sfGFP - ter | BBa_K5299200 https://parts.igem.org/Part:BBa_K5299200 | pDGB3αlpha1 |
| P3.1 - RBS2 - sfGFP - ter | BBa_K5299201 https://parts.igem.org/Part:BBa_K5299201 | pDGB3αlpha1 |
| PJ23119 - RBS1 - sfGFP - ter | BBa_K5299202 https://parts.igem.org/Part:BBa_K5299202 | pDGB3αlpha1 |
| PJ23119 - RBS2 - sfGFP - ter | BBa_K5299203 https://parts.igem.org/Part:BBa_K5299203 | pDGB3αlpha1 |
| OsmY - RBS1 - sfGFP - ter | BBa_K5299204 https://parts.igem.org/Part:BBa_K5299204 | pDGB3αlpha1 |
| OsmY - RBS2 - sfGFP - ter | BBa_K5299205 https://parts.igem.org/Part:BBa_K5299205 | pDGB3αlpha1 |
| BG37 - RBS2 - sfGFP - ter | BBa_K5299012 https://parts.igem.org/Part:BBa_K5299012 | pDGB3αlpha1 |
| BG17 - RBS2 - sfGFP - ter | BBa_K5299013 https://parts.igem.org/Part:BBa_K5299013 | pDGB3αlpha1 |
| BG42 - RBS2 - sfGFP - ter | BBa_K5299014 https://parts.igem.org/Part:BBa_K5299014 | pDGB3αlpha1 |
| BG37 - RBS2 - Τ7 RNAP- ter | BBa_K5299015 https://parts.igem.org/Part:BBa_K5299015 | pDGB3αlpha1 |
| PJ23- RBS2 - T7 RNAP- ter | BBa_K5299016 https://parts.igem.org/Part:BBa_K5299016 | pDGB3αlpha1 |
| T7 pro- RBS2- sGFP- T7 ter | BBa_K5299018 https://parts.igem.org/Part:BBa_K5299018 | pDGB3αlpha2 |
| T7terhyb6 - T7ter pro- RGS1- T7ter pro- T7terhyb6 | BBa_K5299019 https://parts.igem.org/Part:BBa_K5299019 | pDGB3αlpha2 |
Our Level ω constructs (Golden Braid Assembly)
| Part name | Registry Code | GB Vector |
|---|---|---|
| BG37- RBS2- T7pol- ter (a1)- T7pro-sGFP-T7ter(a2) | BBa_K5299030 https://parts.igem.org/Part:BBa_K5299030 | pDGB3omega1 |
| PJ23119 - RBS2 - T7pol- ter (a1)- T7pro-sGFP-T7ter(a2) | BBa_K5299031 https://parts.igem.org/Part:BBa_K5299031 | pDGB3omega1 |
Golden Standard Assembly
Our composite parts were assembled according to the Golden Standard method, which uses the Standard European Vector Architecture (SEVA) and Modular Cloning (MoClo) syntax. With this system, basic parts are combined into transcriptional units, which can be further assembled into complex genetic circuits. This modularity ensures compatibility with existing part libraries, and allows for the efficient creation of genetic circuits with multiple transcriptional units. These composite parts were expressed in P. putida [2]. Below, you will see the composite parts we assembled and tested during this iGEM season using the Golden Standard system.
Our Level 1 constructs (Golden Standard Assembly)
| Part name | Registry Code | GS Vector |
|---|---|---|
| PJ23119 - RBS2 - sfGFP - ter | BBa_K5299203 https://parts.igem.org/Part:BBa_K5299203 | pSEVA23g19[g1] |
| OsmY - RBS2 - sfGFP - ter | BBa_K5299205 https://parts.igem.org/Part:BBa_K5299205 | pSEVA23g19[g1] |
| BG37 - RBS2 - sfGFP - ter | BBa_K5299012 https://parts.igem.org/Part:BBa_K5299012 | pSEVA23g19[g1] |
For the dsRNA production in vitro we designed primers with T7 promoter sequence overhangs in order to create the DNA template we would use for the in vitro transcription. The underlined letters are the T7 promoter sequence.
| Gene target | Name | Sequence | Product size | Tm ℃ | Registry code |
|---|---|---|---|---|---|
| RGS1 | Vd_RGS1-F | TAATACGACTCACTATAGGGA GATCCGTTCCGCTCAGAAGAAC |
266 | 65.3 | BBa_K5299100 https://parts.igem.org/Part:BBa_K5299100 |
| Vd_RGS1-R | TAATACGACTCACTATAGGGAGA AACAGAGCCGTCACCTCTTG |
65.4 | BBa_K5299101 https://parts.igem.org/Part:BBa_K5299101 | ||
| AAC | Vd_AAC-F | TAATACGACTCACTATAGGGAGA TCGGGCGGTGGTATAATGAC |
237 | 65.2 | BBa_K5299102 https://parts.igem.org/Part:BBa_K5299102 |
| Vd_AAC-R | TAATACGACTCACTATAGGGAGAGG GAAAGAAAACGCCTTGCC |
66 | BBa_K5299103 https://parts.igem.org/Part:BBa_K5299103 | ||
| THI20 | Vd_THI20-F | TAATACGACTCACTATAGGGAGAAG GGCAACGTGTACTGGAAG |
247 | 65.4 | BBa_K5299104 https://parts.igem.org/Part:BBa_K5299104 |
| Vd_THI20-R | TAATACGACTCACTATAGGGAGAAAC GAATCTACTGGCACGGG |
65.5 | BBa_K5299105 https://parts.igem.org/Part:BBa_K5299105 |
We evaluated gene expression with RT-qPCR. We designed primers for our target genes, the DCL genes and a housekeeping gene for normalisation.
| Gene target | Name | Sequence | Product size | Tm ℃ | Registry code |
|---|---|---|---|---|---|
| RGS1 | Vd_RGS1-F | AGTCACAGTCACAACCCGAC | 120 | 59.9 | BBa_K5299106 https://parts.igem.org/Part:BBa_K5299106 |
| Vd_RGS1-R | CGTGTGCGTTCTTGTCCAAG | 60 | BBa_K5299107 https://parts.igem.org/Part:BBa_K5299107 | ||
| AAC | Vd_AAC-F | ACTTCGGCATGTACGACTCC | 118 | 59.8 | BBa_K5299108 https://parts.igem.org/Part:BBa_K5299108 |
| Vd_AAC-R | TAAGAAGCGATACCGGCACC | 59.9 | BBa_K5299109 https://parts.igem.org/Part:BBa_K5299109 | ||
| THI20 | Vd_THI20-F | TTCCCGTGCCAGTAGATTCG | 92 | 59.8 | BBa_K5299110 https://parts.igem.org/Part:BBa_K5299110 |
| Vd_THI20-R | ACCCCCAGTTTCACACCAAG | 60.1 | BBa_K5299111 https://parts.igem.org/Part:BBa_K5299111 | ||
| Histone 3 | Vd_His3-F | CAGCCCTGTCGCGTAAAAAC | 115 | 59.4 | BBa_K5299112 https://parts.igem.org/Part:BBa_K5299112 |
| Vd_His3-R | TCTTTCTTCTCGGCAGGGTG | 59.4 | BBa_K5299113 https://parts.igem.org/Part:BBa_K5299113 | ||
| DCL1 | Vd_DCL1-F | TCGCTGGAGTTGCTCTGAAG | 94 | 60 | BBa_K5299114 https://parts.igem.org/Part:BBa_K5299114 |
| Vd_DCL1-R | ACAGCATGACCCCAGAAAGG | 60 | BBa_K5299115 https://parts.igem.org/Part:BBa_K5299115 | ||
| DCL2 | Vd_DCL2-F | AGAACAGCTTTCCTCGAGCC | 113 | 60 | BBa_K5299116 https://parts.igem.org/Part:BBa_K5299116 |
| Vd_DCL2-R | CAAGGCGCTCGTAGTTTGTG | 59.8 | BBa_K5299117 https://parts.igem.org/Part:BBa_K5299117 |
Conclusion
This concludes our Part Collection page. We hope that future teams can reference this page so as to assist them in their own understanding of the competition and the usefulness of a Part Collection page. We also hope that this page can act as inspiration and a way to aid new iGEMers in their own project’s formation.
[1] Sarrion-Perdigones, A., Falconi, E. E., Zandalinas, S. I., Juárez, P., Fernández-del-Carmen, A., Granell, A., & Orzaez, D. (2011). GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PloS one, 6(7), e21622.
[2] Blázquez, B., León, D. S., Torres-Bacete, J., Gómez-Luengo, Á., Kniewel, R., Martínez, I., Sordon, S., Wilczak, A., Salgado, S., Huszcza, E., Popłoński, J., Prieto, A., & Nogales, J. (2023). Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria. Nucleic acids research, 51(19), e98.
[3] Zobel, S., Benedetti, I., Eisenbach, L., de Lorenzo, V., Wierckx, N., & Blank, L. M. (2015). Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida. ACS synthetic biology, 4(12), 1341–1351. https://doi.org/10.1021/acssynbio.5b00058