Team:Aalto-Helsinki/Registry parts
Registry Parts
The biological component of GutLux comprises the AhR-ARNT transcription factor system and the reporter plasmid. In order to make the host organism sense the target metabolites (i.e., kynurenic acid (KYNA) and tryptamine), we have modified them using genetic engineering tools. The host(s) were transformed using plasmids that were designed to express the proteins of the AhR-ARNT Transcription factor system: human Aryl hydrocarbon Receptor (AhR), human Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) and human Aryl hydrocarbon Receptor Interacting Protein (AIP). More information regarding the AhR-ARNT transcription factor system, the reporter plasmid, and our plasmid backbones can be found on the Wet Lab page.
These plasmids were designed extensively using Modular Cloning (MoClo) (Weber et al., 2011) and as such, every single part of the transcriptional unit can be switched for another similar part. This versatility enabled us to compare and test out a few combinations of standard gene regulatory elements to identify the combination that works best for GutLux. Our design is based on the RFC94 standard pioneered by the Boston U iGEM team. Working with MoClo also meant that we were able to design and characterize some basic parts that are compatible with other existing assembly standards such as BBRFC[10] and type IIS RFC[1000].
Through the design of GutLux, we were able to produce 12 unique MoClo parts and all of them are submitted as basic parts to the iGEM parts registry.
T7 Promoter: BBa_K3793000
A Promoter for bacterial expression in T7 systems. Requires a bacterial host expressing T7 polymerase.
Ribosomal Binding Site: BBa_K3793001
Ribosomal Binding Site for E. coli (Elowitz and Leibler, 2000).
T7 Terminator: BBa_K3793003
Transcription terminator for E. coli.
CDS hAhR: BBa_K3793004
A coding sequence for truncated version of the human Aryl hydrocarbon Receptor (AhR) with an N-terminal 6x Histidine tag. Codes for the residues that comprise the bHLH, PAS-A and PAS-B domains (Schulte et al., 2017).
CDS hARNT: BBa_K3793005
A coding sequence for truncated version of the human Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) with an N-terminal 6x Histidine tag. Codes for the residues that comprise the bHLH, PAS-A and PAS-B domains (Schulte et al., 2017).
CDS hAIP: BBa_K3793006
A coding sequence for full-length human Aryl hydrocarbon Receptor Interacting Protein (AIP) with an N-terminal 6x Histidine tag. Codes for the full length protein containing the FKBP52-like domain and the 3 TPR domains. Sequence was adopted from the Uniprot entry of AIP: O00170.
POP6 Promoter: BBa_K3793007
A yeast expression promoter originally from the POP6 gene that encodes for the ribonuclease P/MRP subunit in Saccharomyces cerevisiae (Lee et al., 2015).
SAC6 Promoter: BBa_K3793008
A yeast expression promoter originally from the SAC6 gene that encodes for an actin-bundling protein in S. cerevisiae (Lee et al., 2015).
RNR2 Promoter: BBa_K3793009
A yeast expression promoter originally from the RNR2 gene that encodes for the ribonucleotide-diphosphate reductase (RNR) in S. cerevisiae (Lee et al., 2015).
CYC1 Terminator: BBa_K3793010
A yeast transcription terminator originally from the CYC1 gene that encodes for cytochrome c protein in S. cerevisiae (Lee et al., 2015).
GST tag with enterokinase site: BBa_K3793011
A coding sequence for the Glutathione S-Transferase (GST) from Schistosoma japonicum, with an enterokinase cleavage site (DDDDK) intended for C-terminal tagging. The GST tag can be cleaved using an enterokinase.
We also constructed 2 reporter constructs using the same RFC94 guidelines to express superfolder Green fluorescent Protein (sfGFP) upon metabolite detection by the AhR-ARNT system. These reporter constructs are modular and can function with any promoter in an appropriate expression system. For the purposes of GutLux, we had the CYP1A1 promoter in the reporter construct. These reporter cassettes (with and without the CYP1A1 promoter) are submitted to the registry as composite parts.
sfGFP plug with space for promoter: BBa_K3793012
We have also submitted another composite part made using the T7 Promoter and the RBS:
T7 Promoter and RBS: BBa_K3793002
Here is a table summarizing all the parts team Aalto-Helsinki 2021 has submitted to the iGEM parts registry:
References
1. Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature 2000 403:6767, 403(6767), 335–338. https://doi.org/10.1038/35002125
2. Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS synthetic biology, 4(9), 975–986. https://doi.org/10.1021/sb500366v
3. Schulte, K. W., Green, E., Wilz, A., Platten, M., & Daumke, O. (2017). Structural Basis for Aryl Hydrocarbon Receptor-Mediated Gene Activation. Structure, 25(7), 1025-1033.e3. https://doi.org/10.1016/j.str.2017.05.008
4. Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PloS one, 6(2), e16765. https://doi.org/10.1371/journal.pone.0016765
