Team:Stony Brook/Parts

iGEM SBU 2021

Navigating to Parts

IPTG inducible promoter from Browning et al.

This part is promoter from Lutz and Bujard (1997) and was improved upon by the Groningen 2019 iGEM team. It is a constitutive promoter that is repressed by tetracycline, meaning that it is “ON,” except in the presence of tetracycline. Repression by tetracycline is inhibited by anhydrotetracycline (aTc). Therefore, expression can be regulated by changing the amount of aTc added, making it an inducible promoter.

This is a ribosome binding site from the Anderson RBS family, which is suitable for general protein expression in E. coli.

Browning et al.

This part is the coding sequence for the microcystinase protein (MlrA) originally isolated from Sphingomonas sp. ACM-3962, and codon optimized by the 2019 Aalto-Helsinki iGEM team. The C-terminus of the protein includes a 6X His-tag, connected via a linker, in order to allow for protein purification using Nickel affinity chromatography and detection using Western blotting with anti-His antibodies.

This is a part for use in E. coli that causes transcription termination via a 64 bp stem-loop structure, independent of any protein factors.

See above.

This part codes for a subunit of poly-γ-glutamic acid synthetase from Bacillus licheniformis. It is an anchoring protein which transports protein to the outer membrane. It has been codon optimized for E. coli using the IDT Codon Optimization Tool.

This part codes for a 2 amino acid glycine serine linker.

See above.

This part is a strong constitutive promoter from the Anderson series that has been thoroughly characterized by previous iGEM teams.

This is a ribosome binding site from the Anderson RBS family, which is suitable for general protein expression in E. coli.

This part encodes the catalytic domain of human protein phosphatase-1 (PP1) and includes a C-terminal hemagglutinin epitope tag. It was designed by the 2013 Dundee iGEM team. PP1 is a serine/threonine phosphatase, meaning it dephosphorylates serine and threonine residues on proteins, and regulates many biological processes including cell progression and protein synthesis. The ADDA (3-amino-9-methoxy-2,6,8-trimethyl- 10-phenyl-4,6-decadienoic) site of microcystin-LR binds to PP1, inactivating it and causing hepatotoxic and potentially carcinogenic effects (Zong et al., 2017). This part is being used as one half of the two-hybrid system, modeled after the 2009 Tianjin) iGEM team’s yeast two-hybrid assay for MC-LR detection.

This is a 54 amino acid glycine glycine serine linker, used by the 2019 Grenoble-Alpes iGEM team in their bacterial two-hybrid assay system.

This part encodes for one part of the catalytic domain of adenylyl cyclase from Bordetella pertussis, the other domain being T25. T18 and T25 together catalyze the conversion of ATP to cyclic adenosine monophosphate (cAMP). In our construct, T18 is linked to PP1 in order to ultimately cause cAMP production in the presence of MC-LR. This in turn triggers transcription of the GFP gene, which is downstream of a cAMP-inducible promoter.

This part is a double terminator consisting of parts BBa_B0010 and BBa_B0012. Three different terminators were used for the detection system inserts in order to prevent off-target annealing of the overhangs during HiFi assembly.

See above.

This part encodes for glutathione (GSH), which is an antioxidant that binds to the Mdha (methyl-dehydroalanine) site of MC-LR (Zong et al., 2017). This part is being used as the other half of the two-hybrid system, in addition to PP1, modeled after the 2009 Tianjin iGEM team’s yeast two-hybrid assay for MC-LR detection.

See above.

This part encodes for one part of the catalytic domain of adenylyl cyclase from Bordetella pertussis, the other domain being T18. T18 and T25 together catalyze the conversion of ATP to cyclic adenosine monophosphate (cAMP). In our construct, T25 is linked to GSH in order to ultimately cause cAMP production in the presence of MC-LR. This in turn triggers transcription of the GFP gene, which is downstream of a cAMP-inducible promoter.

This part is the T1 terminator from the rnpB gene of E. coli MG1655.

This part is the promoter from the lac operon, which is repressed by the lacI repressor and induced by cAMP binding to cAMP receptor protein (CRP, also known as catabolite activating protein, or CAP). When cAMP binds to CRP, it causes a conformational change that promotes RNA polymerase II binding to the DNA and induces transcription.

See above.

This part encodes for green fluorescent protein derived from the jellyfish Aequorea victoria. In the presence of MC-LR, the two subunits of adenylyl cyclase will come together and cause cAMP production, leading to induction of the lac promoter and transcription of GFP.

See above.

Browning, D. F., Richards, K. L., Peswani, A. R., Roobol, J., Busby, S. J. W., & Robinson, C. (2017). Escherichia coli “TatExpress” strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway. Biotechnology and Bioengineering, 114(12), 2828–2836. https://doi.org/doi.org/10.1002/bit.26434

Lutz, R., & Bujard, H. (1997). Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic acids research, 25(6), 1203–1210. https://doi.org/10.1093/nar/25.6.1203

Zong, W., Wang, X., Du, Y., Zhang, S., Zhang, Y., & Teng, Y. (2017). Molecular Mechanism for the Regulation of Microcystin Toxicity to Protein Phosphatase 1 by Glutathione Conjugation Pathway. BioMed research international, 2017, 9676504. https://doi.org/10.1155/2017/9676504

Team:Stony Brook/Parts

Navigating to Parts

Parts Overview

TatExpress

pTac promoter (BBa_K3802001)

pTET Inducible Promoter (BBa_K3171173)

RBS (BBa_J61100)

TorA Signal Sequence

4aa Mature TorA Protein

MlrA w/ 6X HisTag (BBa_K1907002)

rrnB T1 terminator (BBa_B0010)

PgsA Anchoring

pTac promoter (BBa_K3802001)

RBS (BBa_J61100)

PgsA (Codon optimized) (BBa_K2963020)

GS Linker (BBa_J18920)

MlrA w/ 6xHis (BBa_K1907002)

rrnB-T1 terminator (BBa_B0010)

Detection system

Promoter (BBa_J23112)

RBS (BBa_J61102)

PP1 (BBa_K1012001)

GGS Linker (BBa_K3128010)

T18 (BBa_K1638004)

rrnBT1-T7Te terminator (BBa_B0015)

Promoter (BBa_J23112)

RBS (BBa_J61102)

GSH (BBa_K2571001)

PP1 (BBa_K1012001)

T25 (BBa_K1638002)

Terminator (BBa_J61048)

Promoter (modified, (BBa_K121011)

RBS (BBa_J61102)

GFP (BBa_E0040)

rrnB-T1 terminator (BBa_B0010)

References