Parts

Parts. Stacks of agar plates on a bench, labelled by hand on sticky notes reading Module 4, Parts Module 1+2+3 and Mixed GRIDS.

Abstract

Thirty-one parts registered on the iGEM Registry this year, and two more in draft: a green-light switch in Bacillus subtilis with a secreted plant protectant behind it.

The collection is built in four levels. Level 0 holds the three protectant proteins with their recoded variants, the seven fusions that add a secretion signal and a His tag, and three regulatory elements from the SubtiToolKit. Level 1 puts a fusion behind a promoter. Nine protectant cassettes sit there, two of them light-responsive, beside the three units that make the switch. Level 2 joins all four modules in one shuttle vector, once for each signal peptide. Four entries carry characterisation, all Western blots of His-tagged ACC deaminase: the constitutive cassettes show the secreted enzyme at about 37 kDa, and the two Level 2 devices were blotted under green and red light at 13 and 16 hours. Seventeen entries carry the build and how it was verified. The synthesised coding sequences carry their design, and every documented entry records where the part was used.

The collection

Contents

On this page

Group

Type

Documentation

33 parts

The collection
Level 2 The whole system 2 parts Both devices join the four modules in one pSTK shuttle vector: green light in, secreted ACC deaminase out. They differ only in the signal peptide that carries the enzyme through the cell wall, and both were verified at their three internal junctions.
ccaSR-Csn-AcdS-HistagThe whole system BBa_2654HAF3 Light-controlled device Characterisation

CompositeThe four modules in one vector: phycocyanobilin from ho1-pcyA, CcaS-m3 to see green light, CcaR to pass the signal on, and PcpcG2-veg driving ACC deaminase out of the cell behind the Csn signal peptide. On in green light, off in red.
ccaSR-SamyQ-AcdS 2.1-HistagThe whole system BBa_26U8UTMH Light-controlled device Characterisation

CompositeThe same device with SamyQ in place of Csn and the recoded AcdS 2.1 as its cargo.
Level 1 Light circuit modules 3 parts Three Level 1 transcription units make the switch: the chromophore the receptor needs, the receptor itself, and the regulator that carries the signal to the promoter. The light-sensing unit is deposited behind PliaG.
PtagC+RBS-Ho1:pcyA-L3S2P21Light circuit modules BBa_26XZYM10 PCB production module Experiment

CompositeTurns the cell's own haem into phycocyanobilin, the chromophore CcaS-m3 binds before it can respond to light.
PliaG-RBS0-ccaS-m3-B0015Light circuit modules BBa_261XH2RS Light sensing module Experiment

CompositeCcaS-m3 held on PliaG. Given phycocyanobilin it autophosphorylates under green light and starts the signal.
Pveg-RBS0-ccaR-B0015Light circuit modules BBa_26BDO8AQ Transduction module Experiment

CompositeCcaR on Pveg. It takes the phosphoryl group from CcaS-m3 and switches PcpcG2-veg on.
Level 1 Green-light cassettes 2 parts The light-responsive half of the system: a protectant behind PcpcG2-veg, switched by the three circuit modules above it.
PcpcG2-veg-EEBD-RBS0-SamyQ-AcdS 2.1-Histag-B0015Green-light cassettes BBa_26DE5E2Q Green-light cassette Experiment

CompositeACC deaminase behind the green-light promoter, carried out of the cell by SamyQ.
PcpcG2-veg-EEBD-RBS0-Csn-AcdS-Histag-B0015Green-light cassettes BBa_26Q3LKZU Green-light cassette Experiment

CompositeThe same cassette with the Csn signal peptide and the wild-type enzyme.
Level 1 Protectant cassettes 8 parts The same cargoes behind the constitutive Pveg. They were built to work secretion and detection out on their own, and they give the light-controlled version a benchmark. The truncated BoPep4 cassette is registered and still in draft.
Pveg-EEBD-RBS0-Csn-AcdS-Histag-B0015Protectant cassettes BBa_26CXI09X Protectant cassette Characterisation

CompositeACC deaminase on the constitutive promoter, secreted with Csn. The Western blots and the bioreactor time course were run on this construct.
Pveg-EEBD-RBS0-SamyQ-AcdS 2.1-Histag-B0015Protectant cassettes BBa_26XVQ3FS Protectant cassette Characterisation

CompositeThe same cassette with SamyQ and the recoded AcdS 2.1.
Pveg-EEBD-RBS0-Csn-LEA14-Histag-B0015Protectant cassettes BBa_26SG0NQQ Protectant cassette Experiment

CompositeLEA14 behind Pveg, secreted with Csn.
Pveg-EEBD-RBS0-SamyQ-LEA14 2.1-Histag-B0015Protectant cassettes BBa_26D5AX7S Protectant cassette Experiment

CompositeLEA14 2.1 behind Pveg, secreted with SamyQ.
Pveg-EEBD-RBS0-SamyQ-BoPep4-Histag-B0015Protectant cassettes BBa_26FSZTWM Protectant cassette Experiment

CompositeThe broccoli elicitor peptide behind Pveg, secreted with SamyQ.
Pveg-EEBD-RBS0-SamyQ-BoPep4 2.1-Histag-B0015Protectant cassettes BBa_269A97I7 Protectant cassette Experiment

CompositeThe same cassette carrying BoPep4 2.1, the first recoding.
Pveg-EEBD-RBS0-SamyQ-BoPep4 2.2-Histag-B0015Protectant cassettes BBa_26IA55UY Protectant cassette Experiment

CompositeThe same cassette carrying BoPep4 2.2, the second recoding.
Pveg-EEBD-RBS0-SamyQ-Bopep4-Histag (15-23)-B0015Protectant cassettes Draft Protectant cassette —

CompositeThe 15 to 23 truncation in the same constitutive cassette, with SamyQ and His6.
Level 0 Secretion fusions 7 parts Level 0 entry parts: one cargo, a signal peptide at the N terminus to take it out of the cell, and a His tag at the C terminus to find it again. Csn is the chitosanase signal, SamyQ the α-amylase signal.
Csn-AcdS-HistagSecretion fusions BBa_26NXOP7S Secretion fusion Experiment

CompositeACC deaminase with the chitosanase signal peptide in front of it and His6 behind.
SamyQ-AcdS 2.1-HistagSecretion fusions BBa_26TOGLX3 Secretion fusion Experiment

CompositeAcdS 2.1 with the α-amylase signal peptide SamyQ and His6.
Csn-LEA14-HistagSecretion fusions BBa_26ZQ1T72 Secretion fusion Experiment

CompositeLEA14 with Csn and His6.
SamyQ-LEA14 2.1-HistagSecretion fusions BBa_26J0GJGZ Secretion fusion Experiment

CompositeLEA14 2.1 with SamyQ and His6.
SamyQ-BoPep4-HistagSecretion fusions BBa_26ILD3SO Secretion fusion Experiment

CompositeBoPep4 with SamyQ and His6.
SamyQ-BoPep4 2.1-HistagSecretion fusions BBa_26PX0UGH Secretion fusion Experiment

CompositeBoPep4 2.1 with SamyQ and His6.
SamyQ-BoPep4 2.2-HistagSecretion fusions BBa_267B69E3 Secretion fusion Experiment

CompositeBoPep4 2.2 with SamyQ and His6.
Level 0 Cargo sequences 8 parts The three proteins the system delivers, each codon-optimised for Bacillus subtilis, with the recodings of their first fifteen codons and one truncation. The truncation is registered and still in draft.
AcdSCargo sequences BBa_26MA70QI ACC deaminase Design

BasicACC deaminase from Pseudomonas putida UW4. It degrades ACC, the precursor of ethylene, so a plant under stress makes less of the hormone that stunts it.
AcdS 2.1Cargo sequences BBa_262YYNPV ACC deaminase Design

BasicThe same enzyme with its first fifteen codons recoded to loosen the mRNA at the translation start.
LEA14Cargo sequences BBa_26LTB0W1 LEA14 protein Design

BasicA late embryogenesis abundant protein from Arabidopsis thaliana (At1g01470); overexpressing it in the plant is reported to raise salt tolerance.
LEA14 2.1Cargo sequences BBa_26IN6G1D LEA14 protein Design

BasicThe same protein with its first fifteen codons recoded.
BoPep4Cargo sequences BBa_265CD14Z Elicitor peptide Design

BasicA 23-residue plant elicitor peptide from broccoli (Brassica oleracea var. italica) that acts on the plant's own defence signalling.
BoPep4 2.1Cargo sequences BBa_26LYCBW0 Elicitor peptide Design

BasicThe peptide with its first fifteen codons recoded.
Bopep4 2.2Cargo sequences BBa_26ERHWWI Elicitor peptide Design

BasicA second recoding of the same peptide.
Bopep4 (15-23)Cargo sequences Draft Elicitor peptide Design

BasicBoPep4 cut back to residues 15 to 23, to ask whether the short form still signals.
Level 0 Regulatory parts 3 parts Three SubtiToolKit elements, registered so that the composites above can cite them.
PtagC+RBSRegulatory parts BBa_26A4MT29 Promoter + RBS —

CompositeA SubtiToolKit promoter and ribosome binding site in one part. It drives the PCB production module.
RBS0Regulatory parts BBa_26OSLRE4 RBS —

BasicThe SubtiToolKit's strong ribosome binding site for Bacillus subtilis.
EEBD-RBS0Regulatory parts BBa_26V6CPEV RBS + blocking device —

CompositeRBS0 behind the Escherichia coli expression blocking device, so a cassette stays quiet while it is cloned in E. coli and expresses once it is in B. subtilis.

Parts we built on

Part iGEM Registry What it does in our constructs
Promoter veg BBa_K143012 Pveg, the constitutive promoter behind every protectant cassette and the transduction module
PliaG BBa_K823000 The weak constitutive promoter the light-sensing unit is deposited behind
PcpcG2 Green Light-Inducible sfGFP BBa_254UKC9E Where the PcpcG2-veg hybrid promoter comes from
ccaSm3* BBa_K3317069 The miniaturised CcaS photoreceptor
Ccar BBa_K5431017 The response regulator that switches the green-light promoter on
ho1-pcyA BBa_K4354012 The two enzymes that make phycocyanobilin from haem
SamyQ BBa_K1074014 The α-amylase signal peptide, 93 bp, in front of five of our fusions
RiboJ BBa_K1679038 Inside EEBD-RBS0, insulating the ribosome binding site
L3S2P21 Terminator BBa_K2675031 The terminator closing the PCB production module
L3S2P21 Synthetic Terminator BBa_K2486008 The same terminator, registered by a second team and cited beside it
double terminator (B0010-B0012) BBa_B0015 The double terminator closing every other transcription unit

References

Ten referencesThe two CcaSR papers the light switch is designed from, the terminator library, the B. subtilis part toolbox and the SubtiToolKit, then the five the protectant team cite for the recoding work.
  1. Castillo-Hair, S. M., Baerman, E. A., Fujita, M., Igoshin, O. A., & Tabor, J. J. (2019). Optogenetic control of Bacillus subtilis gene expression. Nature Communications, 10(1), 3099. https://doi.org/10.1038/s41467-019-10906-6
  2. Haller, D. J., Castillo-Hair, S. M., & Tabor, J. J. (2025). Optogenetic control of B. subtilis gene expression using the CcaSR system. Methods in Molecular Biology, 2840, 1 to 17. https://doi.org/10.1007/978-1-0716-4047-0_1
  3. Chen, Y.-J., Liu, P., Nielsen, A. A. K., Brophy, J. A. N., Clancy, K., Peterson, T., & Voigt, C. A. (2013). Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nature Methods, 10(7), 659 to 664. https://doi.org/10.1038/nmeth.2515
  4. Guiziou, S., Sauveplane, V., Chang, H.-J., Clerté, C., Declerck, N., Jules, M., & Bonnet, J. (2016). A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acids Research, 44(15), 7495 to 7508. https://doi.org/10.1093/nar/gkw624
  5. Caro-Astorga, J., Rogan, M., Malcı, K., Ming, H., Debenedictis, E., James, P., & Ellis, T. (2025). SubtiToolKit: A bioengineering kit for Bacillus subtilis and Gram-positive bacteria. Trends in Biotechnology, 43(6), 1446 to 1469. https://doi.org/10.1016/j.tibtech.2025.02.004
  6. Lorenz, R., Bernhart, S. H., Höner zu Siederdissen, C., Tafer, H., Flamm, C., Stadler, P. F., & Hofacker, I. L. (2011). ViennaRNA Package 2.0. Algorithms for Molecular Biology, 6, 26. https://doi.org/10.1186/1748-7188-6-26
  7. Kazusa DNA Research Institute. Codon usage database: Bacillus subtilis subsp. subtilis str. 168 (taxid:1423). https://www.kazusa.or.jp/codon/
  8. Singh, S., Cornilescu, C. C., Tyler, R. C., Cornilescu, G., Tonelli, M., Lee, M. S., & Markley, J. L. (2005). Solution structure of a late embryogenesis abundant protein (LEA14) from Arabidopsis thaliana, a cellular stress-related protein. Protein Science, 14(10), 2601 to 2609. https://doi.org/10.1110/ps.051579205
  9. Glick, B. R., Penrose, D. M., & Li, J. (1998). A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria. Journal of Theoretical Biology, 190(1), 63 to 68. https://doi.org/10.1006/jtbi.1997.0532
  10. Wang, A., Guo, J., Wang, S., Zhang, Y., Lu, F., Duan, J., Liu, Z., & Ji, W. (2022). BoPEP4, a C-terminally encoded plant elicitor peptide from broccoli, plays a role in salinity stress tolerance. International Journal of Molecular Sciences, 23(6), 3090. https://doi.org/10.3390/ijms23063090