Team:TUDelft/PartsTest
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Our Best Basic Part﹀
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Our Best Composite Part﹀
♥ Name Description Designer ♥ BBa_K2918000 blablablablablablablablablablablabla TU Delft 2019 -
Our Part Collection﹀
Engineering non-model organisms requires discovery of organism-specific parts or re-tuning of parts characterized in model organisms. Our part collection provides modular cloning (MoClo) (Webber et al., 2018) compatible parts that can be used for predictable expression of genes across different bacterial species.
Our collection provides essential parts to overcome the need to identify parts for different bacterial hosts and insulate the parts from variations associated with microbial context.
Identification of origins of replication for different bacterial species is laborious. Although broad host range plasmids can be used across a wide range of bacterial species, they work in specific groups of microorganisms (Jain and Srivastava., 2013). Our toolkit provides all the components (four proteins and two origins of replication) of the Phi29 replication machinery necessary to establish host independent (orthogonal) replication. The advantage of establishing orthogonal replication, is little or no knowledge of bacterial host is needed and higher control of the system can be established.
Non-model bacterial species have a limited collection of promoters (Yang et al., 2018) . Our toolkit provides T7 promoter variants of different strengths which in combination with a T7 RNA polymerase mixed forward loop (UBER system), can be used for gene expression across different bacterial species. In addition, the collection consists of a broad host range promoter (PBHR) based on conserved regions of -10 region and -35 region sequences in E.coli and B.subtilis (Yang et al., 2018) .
To achieve modularity and predictability of synthetic parts, insulation from their genetic context needs to be established. Sequences downstream of the transcription start site (TSS) of promoters end up in transcripts and are shown to significantly affect translational rates (Lou et al., 2012) . Our toolkit consists of ribozymes which when included in 5’UTR sequences, due to their self cleavage properties, cleave any artifacts left behind in the transcripts upstream of the RBS.
Our toolkit contains an RBS (universal RBS) predicted to work using RBS calculator 2.0 across a range of bacterial species.
Codon usage across bacterial species varies. Our toolkit provides a cross-species codon harmonization tool. The tool can be used to generate coding sequences with equal codon usage across a range of organisms.
Our toolkit consists of variants of T7 terminators. Use of these variants reduces chances of homologous recombination between transcriptional units (Temme et al., 2012) .
Behaviour of parts is influenced by microbial context dependent variations (eg. copy number, transcription rates and translation rates) and to overcome the need to re-characterize parts for different bacterial species, an incoherent feed forward loop (iFFL) can be implemented in a genetic circuit (Segall-Shapiro et al., 2018) . To establish an iFFL genetic circuit, TALE (Transcriptional Activator Like Effector molecule) proteins can be used as repressors. Our toolkit contains TALE protein designed to target a specific DNA sequence. Furthermore, we engineered a series of promoters that can be targeted by the TALE protein.
Part Collection
♥ Name Type Description Designer Length BBa_K2918005 Regulatory 0.1 T7 promoter TUDelft2019 23 BBa_K2918006 Regulatory 0.5 T7 promoter TUDelft2019 23 BBa_K2918009 Regulatory 0.5 T7sp1 promoter TUDelft 2019 41 BBa_K2918035 5'UTR BBa_B0032 RBS and RiboJ TUDelft 2019 94 BBa_K2918013 5'UTR BBa_B0032 RBS and SarJ TUDelft 2019 98 ♥ BBa_K2918000 Regulatory Broad Host Range Promoter (PBHR) TUDelft 2019 56 BBa_K2918011 Regulatory PBHRsp1 TUDelft 2019 56 BBa_K2918038 5'UTR Universal RBS and RiboJ TUDelft 2019 85 BBa_K2918012 Ribozyme Ribo J TUDelft 2019 75 BBa_K2918036 5'UTR SarJ and Universal RBS TUDelft 2019 89 BBa_K2918010 Regulatory T7sp1 TUDelft 2019 41 BBa_K2918016 Terminator T7 terminator variant TUDelft 2019 48 BBa_K2918008 Coding Transcription Activator like Effector protein (TALEsp1) TUDelft2019 2640 BBa_K2918014 RBS Universal RBS TUDelft2019 10 BBa_K2918062 Composite TALEsp1 stablized PBHR promoter TUDelft2019 10 ♥ BBa_K2918034 Coding Φ29 DNA polymerase (DNAP/p2) TUDelft2019 1719 BBa_K2918003 Coding Φ29 Double Strand Binding Protein (DSB/p6) TUDelft2019 315 BBa_K2918002 Coding Φ29 Single Stranded Binding Protein (SSB/p5) TUDelft2019 375 ♥ BBa_K2918001 Coding Φ29 Terminal Protein (TP/p3) TUDelft2019 801 ♥ BBa_K2918033 ORI Φ29 Left origin of replication (Ori-L) TUDelft2019 801 ♥ BBa_K2918061 ORI Φ29 Right origin of replication (Ori-R) TUDelft2019 801 ♥ BBa_K2918039 Ribozyme SarJ TUDelft2019 801 -
All parts﹀
♥ Name Type Description Designer Length BBa_K2918005 Regulatory 0.1 T7 promoter TUDelft2019 23 BBa_K2918006 Regulatory 0.5 T7 promoter TUDelft2019 23 BBa_K2918007 Regulatory Wild type T7 promoter TUDelft2019 23 BBa_K2918009 Regulatory 0.5 T7 promoter variant with TALEsp1 binding site TUDelft 2019 41 BBa_K2918035 RBS BBa_B0032 RBS and RiboJ TUDelft 2019 94 BBa_K2918013 RBS BBa_B0032 RBS and SarJ TUDelft 2019 98 ♥ BBa_K2918034 Regulatory Broad host range promoter TUDelft 2019 56 BBa_K2918011 Regulatory Broad host range promoter with TALEsp1 binding site TUDelft 2019 56 BBa_K2918037 Coding Cross-species harmonized GFP TUDelft 2019 678 BBa_K2918061 Other RBS (B0032) adapted for modular cloning with Ribozymes TUDelft 2019 25 BBa_K2918038 RBS RiboJ and universal binding site TUDelft 2019 85 BBa_K2918012 Other Ribozyme J (Ribo J) TUDelft 2019 75 BBa_K2918036 RBS SarJ and universal ribosome binding site TUDelft 2019 89 BBa_K2918010 Regulatory T7 promoter with TALEsp1 binding site TUDelft 2019 41 BBa_K2918016 Terminator T7 terminator variant TUDelft 2019 48 BBa_K2918008 Coding Transcription Activator like Effector protein (TALEsp1) TUDelft2019 2640 BBa_K2918014 RBS Universal RBS TUDelft2019 10 BBa_K2918062 RBS Universal RBS site adapted for modular cloning with ribozymes TUDelft 2019 16 BBa_K2918015 Terminator Wild type T7 terminator TUDelft2019 48 ♥ BBa_K2918000 Coding Φ29 DNA polymerase (DNAP/p2) TUDelft2019 1719 BBa_K2918003 Coding Φ29 Double Strand Binding Protein (DSB/p6) TUDelft2019 315 BBa_K2918002 Coding Φ29 Single Stranded Binding Protein (SSB/p5) TUDelft2019 375 ♥ BBa_K2918001 Coding Φ29 Terminal Protein (TP/p3) TUDelft2019 801 BBa_K2918017 Composite 0.1 T7 promoter - Universal RBS - Φ29 9 TP - WT T7 terminator TUDelft2019 890 BBa_K2918018 Composite 0.5 T7 promoter - Universal RBS - Φ29 TP - T7 terminator TUDelft2019 904 ♥ BBa_K2918019 Composite WT T7 promoter - Universal RBS - Φ29 TP - T7 terminator TUDelft2019 904 BBa_K2918020 Composite 0.1 T7 promoter - Universal RBS - Φ29 DNAP - T7 terminator TUDelft2019 1822 BBa_K2918021 Composite 0.5 T7 promoter - Universal RBS - Φ29 DNAP - T7 terminator TUDelft2019 1822 BBa_K2918022 Composite WT T7 promoter - Universal RBS - Φ29 DNAP - WT T7 terminator TUDelft2019 1899 BBa_K2918023 Composite 0.1 T7 promoter - Universal RBS - Φ29 DSB (p6) - WT T7 terminator TUDelft2019 519 BBa_K2918024 Composite 0.5 T7 promoter - Universal RBS - Φ29 DSB (p6) - WT T7 terminator TUDelft2019 519 BBa_K2918025 Composite WT T7 promoter - Universal RBS - Φ29 DSB (p6) - WT T7 terminator TUDelft2019 519 BBa_K2918026 Composite 0.1 T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator TUDelft2019 579 BBa_K2918027 Composite 0.5 T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator TUDelft2019 579 BBa_K2918028 Composite WT T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator TUDelft2019 579 BBa_K2918030 Composite WT T7 promoter - Universal RBS - GFP - T7 terminator TUDelft2019 811 BBa_K2918031 Composite 0.1 T7 promoter - Universal RBS - GFP - T7 terminator TUDelft2019 811 BBa_K2918032 Composite 0.5 T7 promoter - Universal RBS - GFP - T7 terminator TUDelft2019 811 References
- Jain, A. and P. Srivastava (2013). "Broad host range plasmids." FEMS Microbiology Letters 348(2): 87-96.
- Segall-Shapiro, T. H., et al. (2018). "Engineered promoters enable constant gene expression at any plasmid copy number in bacteria." Nature Biotechnology 36: 352.
- Yang, S., et al. (2018). "Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology." ACS Synthetic Biology 7(1): 287-291.
- 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.
- Lou, C., Stanton, B., Chen, Y., Munsky, B., & Voigt, C. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology, 30(11), 1137-1142. doi: 10.1038/nbt.2401
- Temme, K., Hill, R., Segall-Shapiro, T., Moser, F., & Voigt, C. (2012). Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic Acids Research, 40(17), 8773-8781. doi: 10.1093/nar/gks597