UnivLyon1-INSALyon

Logo BIO Snare

Materials


All components used for the construction of BIO Snare are listed here:

If you want to know more about the material we used, click on the buttons below.

For our project, we have used both bacterial and yeast cells.

Bacterial strains

Name Source of Reference
Escherichia coli DH5α Laboratory collection
Komagataeibacter rhaeticus Gilbert, C. et al. (2021)

Yeast cells

Name Phenotype Source or Reference
Saccharomyces cerevisiae BY4741 URA3- Laboratory collection - SMAL team

For our project, we have used different plasmidic vectors:

Plasmid name Characteristic Source or Reference
pOpen_V3_promoteurADH1 colE1, AmpR iGEM
Plasmid A colE1, AmpR, URA3 5’ homology, pGAP, α-factor secretion signal v1, fwYellow, ScCBD’v1, TDH1 terminator This study
Plasmid B colE1, AmpR P2A, α-factor secretion signal v2, cp19k, MaSp1, ScCBD’v2 This study
Plasmid C colE1, AmpR, URA+ URA3 5’ homology, URA3 3’ homology, pGAP, α-factor secretion signal v1, fwYellow, ScCBD’v1, TDH1 terminator This study
Plasmid D colE1, AmpR, URA+ URA3 5’ homology, URA3 3’ homology, pGAP, α-factor secretion signal v1, fwYellow, ScCBD’v1, TDH1 terminator, P2A, α-factor secretion signal v2, cp19k, MaSp1, ScCBD’v2 This study
Plasmid Venus/mRuby colE1, AmpR, URA+ URA3 5’ homology, URA3 3’ homology, pGAP, α-factor secretion signal v1, venus, ENO1 terminator, ADH1 promotor, AGA2, TDH1 terminator, mRuby2, 6xHis This study

For our project, we have used different primers:

Name 5’-3’ Sequence Purpose
iGEM 1 TTACCCTGAACGGCCGATCGTGAC
GGTTTCCTTGAAATTTTTTTGATTCG 		used with iGEM17 to verify the insertion of the fwYellow-Bioglue fragment in the BY4741 S. cerevisiae strain.
iGEM 2 TGGCCTTTTGCTCACATGTGATAA
GCTTCTCGAGTCTTTATATTTACA
TGCTAAAAATGGGCTAC 		used with iGEM8 primer on the D plasmid to verify the insertion of the bioglue fragment in the linearised C plasmid.
iGEM 3 - forward ATGCACCGTAGGTGGCTCAACTGG
TTCTGGGGCTACTAACTTTAG 		used with iGEM4 primer to amplify and insert MaSp1 from plasmid pMaSp1 into plasmid C to obtain plasmid D
iGEM 4 - reverse TCAAGATTGCTTTATGGGTCTCCAA
GCTTTTAACCAGTTGACCCTC 		used with iGEM3 primer to amplify and insert MaSp1 from plasmid pMaSp1 into plasmid C to obtain plasmid D
iGEM 5 - reverse AGTTGAGCCACCTACGGTGCAT used with iGEM6 primer to linearize the C plasmid in order to insert Cp19k_MaSp1 fragment in it by cloning.
iGEM 6 - forward CGAGACCCATAAAGCAATCTTGA used with iGEM5 primer to linearize the C plasmid in order to insert Cp19k_MaSp1 fragment in it by cloning.
iGEM 7 - reverse GGTCTCGCGGAATCATTGCAGCACTGG used with iGEM8 primer to remove an unexpected BsaI restriction enzyme site in the C plasmid by IVA PCR.
iGEM 8 - forward AATGATTCCGCGAGACCCACGCTC used with iGEM7 primer to remove an unexpected BsaI restriction enzyme site in the C plasmid by IVA PCR. iGEM8 is also used with iGEM2 primer on the D plasmid to verify the insertion of the bioglue fragment in the linearised C plasmid.
iGEM 9 - reverse ATCGATTCCTCTCTTATCCAGAGAG used with iGEM11 primer to linearise the C plasmid in order to insert the Venus/Ruby fragment in it by cloning.
iGEM 11 - forward ACCGGTATAAAGCAATCTTGATGAGG used with iGEM9 primer to linearise the C plasmid in order to insert the Venus/Ruby fragment in it by cloning.
iGEM 12 - forward TATCCTTTTGTTGTTTCCGGGTGTAC used with iGEM13 primer to amplify the pADH1 promoter (from the 2024 iGEM distribution kit) in order to insert it in the linearised C plasmid by cloning.
iGEM 13 - reverse AGTTGATTGTATGCTTGGTATAGC used with iGEM12 primer to amplify the pADH1 promoter (from the 2024 iGEM distribution kit) in order to insert it in the linearised C plasmid by cloning.
iGEM 16 GGCGGATTACTACCGTTGC iGEM16 primer anneals to the URA region of BY4741 S. cerevisiae strain. This primer used with iGEM2 to verify the insertion of the fwYellow-Bioglue fragment in the yeast.
iGEM 17 GGATTTGGTTAGATTAGATATGGTTTCTC iGEM17 primer anneals to the URA region of BY4741 S. cerevisiae strain. This primer used with iGEM1 to verify the insertion of the fwYellow-Bioglue fragment in the yeast.

For our project, we have used different promoters:

Promotor name Characteristics 5' to 3' sequence Length
GAP Strong constitutive promoter of yeasts TCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAAC
TTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACA
TGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACA
TCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGC
TTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCAC
CAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAG
GCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACAC
AAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACAC
CTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTC
CCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTA
GGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCT
TTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAA		 667 bp
ADH1 Strong constitutive promoter of yeasts ATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACC
AAACCCATACATCGGGATTCCTATAATACCTTCGTTGGACTCCCTAACATGTAGGTGGCGGA
GGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCT
AAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAAT
ACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTT
TTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTT
TTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAATGATGGAAGT
CACTAAAGGAAAAAATTAACGACAAAGACAGCACC
AACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCC
TTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTC
CTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAAGTATA
AATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCC
TTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACT		 705 bp

For our project, we have used different terminators:

Terminator name 5' to 3' sequence Length
TDH1 ATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAAT
ATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGAT
GACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAA
GATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCA
ATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGT
CGGCGGTTAAAATATATTACCCTGAACG 		234 bp
ENO1 AGCTTTTGATTAAGCCTTCTAGTCCAAAAAACACGTTTTTTTGT
CATTTATTTCATTTTCTTAGAATAGTTTAGTTTATTCATTTTAT
AGTCACGAATGTTTTATGATTCTATATAGGGTTGCAAACAAGCA
TTTTTCATTTTATGTTAAAACAATTTCAGGTTTACCTTTTATTC
TGCTTGTGGTGACGCGTGTATCCGCCCGCTCTTTTGGTCACCC
ATGTAT 		225 bp

For our project, we have used different kits:

  • Microplate readers: M200 Pro, TECAN
  • Thermocycler: Applied Biosystems™ A2481, fisher scientific
  • Clonage: NEBuilder HIFI DNA Assembly Cloning kit (ref : E5520S)
  • Minipreparation of plasmid DNA: Kit « Nucleospin Plasmids » Macherey-Nagel® (ref: 740588.250)
  • Restriction enzymes: XhoI (R0146S), KpnI-HF (R3142S) et XbaI (R0145S)
  • Clean-up gel extraction (DNA purification): Kit « PCR Clean-up gel extraction » Macherey-Nagel®
  • PCR on colonies: Master Mix PCR DreamTaq Green (2X)
  • Fragment amplification: PrimeSTAR max mastermix (2X)
  • DNA Assembly Mixes: NEBuilder® HiFi DNA Assembly Master Mix (ref: NEB #E2621S)
  • Competent Cells: NEB 5-alpha Competent E.Coli** (ref: NEB #C29871)
  • DNA Ligase: Hi-T4TM DNA Ligase (ref: NEB #M2622S)
  • Quick-Load® Purple 1kb Plus DNA Ladder (ref: NEB #N0550S)
  • Q5® High-Fidelity 2X Master Mix (ref: NEB #M0492S)
  • Wizard® Plus SV Minipreps DNA Purification Systems (250 preps) (ref: Promega #A1460)
  • Wizard® DNA Clean-Up system (100 preps) (ref: Promega #A7280)
  • Wizard® SV Gel and PCR Clean-Up System (50 preps) (ref: Promega #A9281)

[1] Ye, L. et al. (2023). A bioinspired synthetic fused protein adhesive from barnacle cement and spider dragline for potential biomedical materials. International Journal of Biological Macromolecules 253, 127125.

[2] Gilbert, C. et al. (2021). Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat. Mater. 20, 691–700.

[3] Liu, Z. et al. (2017).Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7, 2193.

[4] Brake, A. J. et al. a-Factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae.

[5] Liljeruhm, J. et al. (2018). Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. J Biol Eng 12, 8.

[6] Ma, C. et al. (2021). Ultra-strong bio-glue from genetically engineered polypeptides. Nat Commun 12, 3613.