Team:TUDelft/Parts
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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 consists of 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 & 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 portable expression systems, can be used for gene expression across different bacterial species. In addition, the collection consists of a broad host range promoter (PBHRsp1) based on conserved regions of -10 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 across a range of bacterial species using RBS calculator 2.0. We have demonstrated the working of the universal RBS in E. coli and P. putida.
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).
Behavior of parts is influenced by microbial context dependent variations (eg. copy number, transcriptional rates and translational rates). 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, Transcriptional Activator Like Effector (TALE) proteins can be used as repressors. Our toolkit contains the gene to express TALE protein which is 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 ♥ BBa_K2918034 Coding Φ29 DNA polymerase (DNAP/p2) ♥ BBa_K2918001 Coding Φ29 Terminal Protein (TP/p3) BBa_K2918002 Coding Φ29 Single Stranded Binding Protein (SSB/p5) BBa_K2918003 Coding Φ29 Double Stranded Binding Protein (DSB/p6) BBa_K2918033 Other Φ29 Left origin of replication (OriL) BBa_K2918061 Other Φ29 Right origin of replication (OriR) ♥ BBa_K2918029 Composite OriL-GFP-Kan-OriR BBa_K2918005 Regulatory Weak T7 promoter BBa_K2918006 Regulatory Medium T7 promoter BBa_K2918009 Regulatory Medium T7sp1 promoter BBa_K2918010 Regulatory T7sp1 promoter ♥ BBa_K2918054 Regulatory PBHR sp1 promoter BBa_K2918016 Other T7 terminator variant BBa_K2918014 RBS Universal RBS BBa_K2918012 Other RiboJ BBa_K2918039 Other SarJ BBa_K2918013 RBS BBa_B0032 RBS and SarJ BBa_K2918035 RBS BBa_B0032 RBS and RiboJ BBa_K2918036 RBS Universal RBS and SarJ BBa_K2918038 RBS Universal RBS and RiboJ BBa_K2918008 Coding TALEsp1 ♥ BBa_K2918050 Composite Cross-species based iFFL ♥ BBa_K2918040 Composite T7 promoter based optimized iFFL BBa_K2918046 Composite T7 promoter based iFFL BBa_K2918048 Composite Medium T7 promoter based iFFL -
Our Favorite Basic Part﹀
Our favorite basic part is PBHRsp1 promoter, BBa_K2918054. We successfully demonstrated its function in E. coli and P. putida. Furthermore, it contains a binding site for TALE repressor protein, facilitating its implementation in iFFL genetic circuits. Hence, this promoter can be used for controllable gene expression across different bacterial species.
Figure 1: The functioning of our favorite part, PBHRsp1 promoter, BBa_K2918054, has been confirmed qualitatively in P. putida -
Our Favorite Composite Part﹀
Our favorite composite part is a T7 promoter based optimized iFFL, BBa_K2918040. This part has been demonstrated to insulate gene expression from variations in T7 RNAP concentrations and promoter strengths. Furthermore, it is compatible with portable expression systems. Thereby, setting the basis for the development of universal expression systems.
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All Parts﹀
♥ Name Type Description BBa_K2918000 Regulatory PBHR ♥ BBa_K2918001 Coding Φ29 Terminal Protein (TP/p3) BBa_K2918002 Coding Φ29 Single Stranded Binding Protein (SSB/p5) BBa_K2918003 Coding Φ29 Double Stranded Binding Protein (DSB/p6) BBa_K2918005 Regulatory Weak T7 promoter BBa_K2918006 Regulatory Medium T7 promoter BBa_K2918007 Regulatory Wild type T7 promoter BBa_K2918008 Coding Transcription Activator like Effector protein (TALEsp1) BBa_K2918009 Regulatory Medium T7sp1 promoter BBa_K2918010 Regulatory T7sp1 promoter BBa_K2918011 Regulatory PBHR sp1 promoter BBa_K2918012 Other RiboJ BBa_K2918013 RBS BBa_B0032 RBS and SarJ BBa_K2918014 RBS Universal RBS BBa_K2918015 Terminator Wild type T7 terminator BBa_K2918016 Terminator T7 terminator variant BBa_K2918017 Composite Weak T7 promoter - Universal RBS - Φ29 TP - WT T7 terminator BBa_K2918018 Composite Medium T7 promoter - Universal RBS - Φ29 TP - T7 terminator BBa_K2918019 Composite WT T7 promoter - Universal RBS - Φ29 TP - T7 terminator BBa_K2918020 Composite Weak T7 promoter - Universal RBS - Φ29 DNAP - T7 terminator BBa_K2918021 Composite Medium T7 promoter - Universal RBS - Φ29 DNAP - T7 terminator BBa_K2918022 Composite WT T7 promoter - Universal RBS - Φ29 DNAP - WT T7 terminator BBa_K2918023 Composite Weak T7 promoter - Universal RBS - Φ29 DSB (p6) - WT T7 terminator BBa_K2918024 Composite Medium T7 promoter - Universal RBS - Φ29 DSB (p6) - WT T7 terminator BBa_K2918025 Composite WT T7 promoter - Universal RBS - Φ29 DSB (p6) - WT T7 terminator BBa_K2918026 Composite Weak T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator BBa_K2918027 Composite Medium T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator BBa_K2918028 Composite WT T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator ♥ BBa_K2918029 Composite OriL-GFP-Kan-OriR BBa_K2918030 Composite WT T7 promoter - Universal RBS - GFP - T7 terminator BBa_K2918031 Composite Weak T7 promoter - Universal RBS - GFP - T7 terminator BBa_K2918032 Composite Medium T7 promoter - Universal RBS - GFP - T7 terminator BBa_K2918033 Other Φ29 Left origin of replication (OriL) ♥ BBa_K2918034 Coding Φ29 DNA polymerase (DNAP/p2) BBa_K2918035 RBS BBa_B0032 RBS and RiboJ BBa_K2918036 RBS SarJ and Universal RBS BBa_K2918037 Coding Cross-species harmonized eGFP BBa_K2918038 RBS RiboJ and Universal RBS BBa_K2918039 Other SarJ ♥ BBa_K2918040 Composite T7 promoter based optimized iFFL BBa_K2918041 Coding Kanamycin resistance gene BBa_K2918043 Composite T7sp1 - RiboJ_UniRBS - hGFP - T7 terminator BBa_K2918044 Composite T7sp1 - RiboJ_UniRBS - jGFP - T7 terminator BBa_K2918045 Composite Pbhrsp1 - RiboJ_UniRBS - jGFP - T7 terminator BBa_K2918046 Composite T7 promoter based iFFL BBa_K2918048 Composite Medium T7 promoter based iFFL BBa_K2918049 Composite Pbhrsp1_v2 - RiboJ_URBS - jGFP - T7 terminator ♥ BBa_K2918050 Composite Cross-species based iFFL BBa_K2918051 Composite Pbhr_sp2 - RiboJ_URBS - jGFP - T7 terminator BBa_K2918052 Coding eGFP BBa_K2918053 Regulatory T7 promoter (reverse complement) ♥ BBa_K2918054 Regulatory PBHR sp1 promoter BBa_K2918055 Regulatory PBHR sp2 promoter BBa_K2918061 Other Φ29 Right origin of replication (OriR) BBa_K2918062 Composite Universal RBS (adapted for modular cloning with ribozymes)
References
- Jain, A. & P. Srivastava (2013). "Broad host range plasmids." FEMS Microbiology Letters 348(2): 87-96.
- 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.
- Segall-Shapiro, T. H., et al. (2018). "Engineered promoters enable constant gene expression at any plasmid copy number in bacteria." Nature Biotechnology 36: 352. 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.
- 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.
- Yang, S., et al. (2018). "Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology." ACS Synthetic Biology 7(1): 287-291.