Team:Sherbrooke/PartsDesign
Parts Design
Parts design
An overview of all the parts’ design is shown here. For more details about the construction of the parts, you may refer to the experiments section
BBa_K1744000
A RBS was selected through rational design and added with a primer to pVCR94's toxin (vcrx028) coding sequence and it was cloned in the plasmid pBAD30 (using the site EcoRI). The part itself is a region of the resulting plasmid, from araC to bla.
Back to top
BBa_K1744001
The part was constructed using a kanamycin resistance gene from the plasmid pOK12 (available commercially) and amilCP from BBa_K592009. The promoter and RBS P1U8 (from Mutalik et al. (Nature 2013)) were synthesized and joined to amilCP.
Back to top
BBa_K1744002
The part was constructed using araC and PBAD of pBAD30 (commercially available), a RBS selected through rational design, the toxin vcrx028 from the conjugative plasmid pVCR94, P1U8 that are a strong constitutive promoter and a strong RBS from Mutalik et al. (Nature 2013), amilCP, a chromoprotein isolated from Acropora millepora that was BioBrick adapted in E.coli by a previous IGEM team (see BBa_K592009) and KanR (Aph(3’)-I (aminoglycoside3’-phosphotransferase)) from pOK12 (it is a truncated version). In practice, this part was built from two previously submitted parts of our team: BBa_K1744000 and BBa_K1744001.
Back to top
Overview
First of all, different toxin CDS were cloned (separately) in pBAD30 to obtain different kill switches. Different tests were done with those (see the experiments section) to choose the best one. And that was the one with vcrx028. We then took the part of the plasmid pBAD30-vcrx028 from araC to ampR (BBa_K1744000) and cloned it in pSB1C3. Separately, we also joined a known iGEM part, BBa_K592009, that is amilCP with aph, a kanamycin resistance gene, and cloned those 2 genes (BBa_K1744001) in pSB1C3. Then, we took a region of BBa_K1744000 and joined it with BBa_K1744001 to obtain BBa_K1744002.
Back to top
Primers
| Primer Name | Primer sequence | Anneal Tm (C) | Anneal to | Goal |
|---|---|---|---|---|
| vcrx028-pBAD30-F | TCTCCATACCCGTTTTTTTGGGCTAGCGTAGG AGGCAAAAATGTGGGTCATCGAGACAA | 50 | pVCR94 vcrx028 | Build pBAD30-vcrx028 |
| vcrx028-pBAD30-R | ACTCTAGAGGATCCCCGGGTACCGAGCTCGTC ACTCCTTCTTCAATTTATCCAAG | 50 | pVCR94 vcrx028 | Build pBAD30-vcrx028 |
| ccdB-pBAD30-F | TCCATACCCGTTTTTTTGGGCTAGCGTAGGAG GCAAAAATGCAGTTTAAGGTTTACACCT | 50 | ptac-ccdb-casette(gBlock) | Build pBAD30-ccdB |
| ccdB-pBAD30-R | ACTCTAGAGGATCCCCGGGTACCGA GCTCGTTATATTCCCCAGAACATCAGGT | 50 | ptac-ccdb-casette(gBlock) | Build pBAD30-ccdB |
| mosT-pBAD30-F | CCCGTTTTTTTGGGCTAGCGTAGGAG GCAAAAGTGAACAACATAATGGATAAAGATAGCC | 50 | pSXT | Build pBAD30-mosT |
| mosT-pBAD30-R | ACTCTAGAGGATCCCCGGGTACCGAG CTCGTTACTCGGCGTTTGCGTT | 50 | pSXT | Build pBAD30-mosT |
| mazF-pBAD30-F | TTCTCCATACCCGTTTTTTTGGGCTAGCG TAGGAGGCAAAAATGGTCTCACGCTATGTCC | 50 | gBlock-FG1 | Build pBAD30-mazF |
| mazF-pBAD30-R | ACTCTAGAGGATCCCCGGGTACCGAGC TCGTTAGCCGATCAGGACATTAATTTTAG | 50 | gBlock-FG1 | Build pBAD30-mazF |
| vcrx028-KanR-del-F | TTCTGTTGCCAGCATTTGGTCAAGAGTT CTTGCCATAACGGTGTAGGCTGGAGCTGCTTC | 55 | pKD4 | Remove vcrx028 from pVCR94 |
| vcrx028-KanR-del-R | TTTTAGTCACATGACTACTTACTGACATT TCGGCCCCCATATGGGAATTAGCCATGGTCC | 55 | pKD4 | Remove vcrx028 from pVCR94 |
| vcrx027-del-F | AGCAAGAGAAGTAATACGCCCCATAACGG GGCGTATCGAGATTGTCTGATTCGTTACCAA | 50 | pBAD30-vcrx028 | Remove vcrx027 (anti-toxin) and previous KanR from pVCR94 |
| vcrx027-del-R | TTTTAGTCACATGACTACTTACTGACATT TCGGCCCCCATTCGAGTAAACTTGGTCTGACAG | 50 | pBAD30-vcrx028 | Remove vcrx027 (anti-toxin) and previous KanR from pVCR94 |
| vcrx027-28-clean-del-5'-F | TCACTGGGTTTGGTACTTTC | 50 | pVCR94 upstream vcrx027 | Clean deletion homology and cassette assembly |
| vcrx027-28-clean-del-5'-R | TTTTAGTCACATGACTACTTACTGACATTT CGGCCCCCATCGATACGCCCCGTTATGGG | 50 | pVCR94 upstream vcrx027 | Clean deletion homology and cassette assembly |
| vcrx027-28-clean-del-3'-F | GTATATGGGGCGACTGCACGTATGGGGGC CGAAATGTCAG | 55 | pVCR94 downstream vcrx028 | Clean deletion homology and cassette assembly |
| vcrx027-28-clean-del-3'-R | CCTGTTGAATGCCAAGCGG | 55 | pVCR94 downstream vcrx028 | Clean deletion homology and cassette assembly |
| vcrx027-28-del-verif-F | ACATTCCTGGGTTCAAAGC | 50 | pVCR94 vcrx027-28 region | Deletion verification primer |
| vcrx027-28-del-verif-R | GATTATTTGCCCGCTCGTG | 50 | pVCR94 vcrx027-28 region | Deletion verification primer |
| pBAD30-vcrx028-igem-ship-F | CGCTAAGGATGATTTCTGGAATTCGCGGCCGC TTCTAGAGCAATTGTCTGATTCGTTACC | 50 | pBAD30-vcrx028 | Build BBa_K1744000 + BBa_K1744002 |
| pBAD30-vcrx028-igem-ship-R | CTTGCCCTTTTTTGCCGGACTGCAGCGGCCGC TACTAGTAGAGTAAACTTGGTCTGACAG | 50 | pBAD30-vcrx028 | Build BBa_K1744000 |
| kanR-amilCP-igem-ship-F1 | CGCTAAGGATGATTTCTGGAATTCGCGGCCGC TTCTAGAGTTAGAAAAACTCATCGAGCA | 50 | pOK12 for KanR | Build BBa_K1744001 |
| kanR-amilCP-igem-ship-R1 | TGAAATTTCCATTGCACGCAAACCTGTGGTCGC CTAATAAATGAGCCATATTCAACGGGA | 50 | pOK12 for KanR | Build BBa_K1744001 |
| kanR-amilCP-igem-ship-F2 | TTATTAGGCGACCACAGGTT | 50 | gBlock_IGEM_2015_2 | Build BBa_K1744001 |
| kanR-amilCP-igem-ship-R2 | CTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTA CTAGTATTTACAGCTAGCTCAGTCCT | 50 | gBlock_IGEM_2015_2 | Build BBa_K1744001 |
| vcrx028-kan-amilCP-IGEM-ship-R1 | TTTCAAAGCTAGCATAATACCTAGGACTGAGCTA GCTGTAAACTAGAGGATCCCCGGGTA | 50 | pBAD30-vcrx028 | Build BBa_K1744002 plasmid + genomic cassette assembly |
| vcrx028-kan-amilCP-IGEM-ship-F2 | TTTACAGCTAGCTCAGTCCTAG | 50 | BBa_K1744001 | Build BBa_K1744002 plasmid + genomic cassette assembly |
| vcrx028-kan-amilCP-IGEM-ship-R2 | CTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTA CTAGTATTAGAAAAACTCATCGAGCA | 50 | BBa_K1744001 | Build BBa_K1744002 |
| kanR-amilCP-lacZ-ins-F | TTATTTTTGACACCAGACCAACTGGTAATGGTA GCGACCGTTTACAGCTAGCTCAGTCCT | 50 | BBa_K1744001 | BBa_K1744001 insertion in lacZ |
| kanR-amilCP-lacZ-ins-R | GGATGGTGGCGCTGGATGGTAAGCCGCTGGCAAG CGGTGATTAGAAAAACTCATCGAGCA | 50 | BBa_K1744001 | BBa_K1744001 insertion in lacZ |
| vcrx028-kan-amilCP-lacZ-ins-F | TTATTTTTGACACCAGACCAACTGGTAATGGTAG CGACCGCAATTGTCTGATTCGTTACC | 50 | BBa_K1744002 | BBa_K1744002 insertion in lacZ |
| vcrx028-kan-amilCP-lacZ-ins-R | GGATGGTGGCGCTGGATGGTAAGCCGCTGGCAA GCGGTGATTAGAAAAACTCATCGAGCA | 50 | BBa_K1744002 | BBa_K1744002 insertion in lacZ |
| lacZ-homology-ext-F | C*G*CGAAATACGGGCAGACATGGCCTGCCCGG TTATTATTATTTTTGACACCAGACCAACT | 55 | lacZ insertion cassette | Insertion in lacZ homology extension |
| lacZ-homology-ext-R | G*A*ATTCTCATGTTTGACAGCTTATCATCGG AGCTCCTGCACTGGATGGTGGCGCTGGATG | 55 | lacZ insertion cassette | Insertion in lacZ homology extension |
| 5'_pVCR94_del_verif_F | ACATTCCTGGGTTCAAAGC | 50 | pVCR94 vcrx26 region | Verification of the insertion of BBa_K1744000 in pVCR94 |
| 3'_pVCR94_del_verif_R | CATGACTACTTACTGACATTTCGGC | 50 | pVCR94 vcrx29 region | Verification of the insertion of BBa_K1744000 in pVCR94 |
| 5'_pVCR94_del_verif_R | CGCCAGCAGTTAGGGATTAG | 55 | BBa_K1744000 | Verification of the insertion of BBa_K1744000 in pVCR94 |
| 3'_pVCR94_del_verif_F | GGCGGATAAAGTTGCAGGAC | 55 | BBa_K1744000 | Verification of the insertion of BBa_K1744000 in pVCR94 |
| killswitch_ins_LacZ_F | TTATTTTTGACACCAGACCAACTGGTAATGG TAGCGACCGATTGTCTGATTCGTTACCAA | 50 | pBAD30 | Toxin-in-LacZ-phenotype |
| killswitch_ins_LacZ_R | GGATGGTGGCGCTGGATGGTAAGCCGCTGGCA AGCGGTGAGAGTAAACTTGGTCTGACAG | 50 | pBAD30 | Toxin-in-LacZ-phenotype |
| lacZ-verif-F | TACTAGTAGCGGCCGCTGC | 55 | Upstream of lacZ | Verification of BBa_K1744001 insertion in LacZ |
| lacZ-verif-R | GAGCTCCTGCACTGGATGGT | 60 | lacZ | Verification of BBa_K1744001 insertion in LacZ |
| amilCP-R | TTATTAGGCGACCACAGGTT | 55 | amilCP | Verification of BBa_K1744001 insertion in LacZ |
Back to top
References
- Datsenko KA, Wanner BL, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc Natl Acad Sci USA 97, 6640(5), 2000.
- Mutalik K V, Guimaraes C J, Cambray G, Mai QA, Christoffersen MJ, Martin L, Yu A, Lam C, Rodriguez C, Bennett G, Keasling D J, Endy D & Arkin P A, Quantitative estimation of activity and quality for collections of functional genetic elements, Nature methods, 10(4), 2013.
- Carraro N, Matteau D, Luo P, Rodrigue S, Burrus V, The Master Activator of IncA/C Conjugative Plasmids Stimulates Genomic Islands and Multidrug Resistance Dissemination. PLoS Genet 10(10), 2014
- Carraro N, Sauve M, Matteau D, Lauzon G, Rodrigue S, et al. (2014) Development of pVCR94DX from Vibrio cholerae, a prototype for studying multidrug resistant IncA/C conjugative plasmids. Front Microbiol 5(44), 2014
- Reddy TR, Kelsall EJ, Fevat LMS, Munson SE, Cowley SM, Differential Requirements of Singleplex and Multiplex Recombineering of Large DNA Constructs, PLoS ONE 10(5), 2015.
Back to top