Part:BBa_K2918038
RiboJ and Universal RBS
Ribosome Binding Site which is functional in both gram positive and gram negative bacteria along with ribozyme (RiboJ).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
The part has been confirmed by sequencing and has no mutations.
Usage and Biology
Many promoter parts contain sequences downstream of the Transcription Start Site (such as operators or assembly fusion sites) resulting in extra unintended sequences in the transcript. These additional sequences are shown to significantly affect gene expression levels, disrupting the modularity and predictability of synthetic parts (Lou et al., 2012).Therefore, to insulate the translation rates of the part from the use of different promoters, ribozymes can be used for their self cleavage properties and remove these sequences upstream of mRNA. By the inclusion of ribozymes in 5’ UTR parts, outputs of genetic circuits will be insulated from genetic context. This BioBrick consists of RiboJ and universal ribosome binding site. The universal RBS has been shown to work in E. coli, B. subtilis and S. cerevisiae (Yang et al., 2017).
Strain Construction
The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective Modular Cloning (MoClo) compatible 5'UTR overhangs. For this particular RBS, the downstream overhang 'AATG' can be modified to 'ATG' since the RBS sequence ends with adenine. This design choice will allow for seamless cloning between the 5'UTR and coding sequence leaving no part-junction sequences. The RBS was then cloned in a level 0 MoClo backbone pICH41246 and the sequence was confirmed by sequencing. The cloning protocol can be found in the modular cloning section below.
Modular Cloning
Modular Cloning (MoClo) is a system which allows for efficient one pot assembly of multiple DNA fragments. The MoClo system consists of Type IIS restriction enzymes that cleave DNA 4 to 8 base pairs away from the recognition sites. Cleavage outside of the recognition site allows for customization of the overhangs generated. The MoClo system is hierarchical. First, basic parts (promoters, UTRs, CDS and terminators) are assembled in level 0 plasmids in the kit. In a single reaction, the individual parts can be assembled into vectors containing transcriptional units (level 1). Furthermore, MoClo allows for directional assembly of multiple transcriptional units. Successful assembly of constructs using MoClo can be confirmed by visual readouts (blue/white or red/white screening). For the protocol, you can find it here.
Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2.
| Level | Basic/Composite | Type | Enzyme |
|---|---|---|---|
| Level 0 | Basic | Promoters, 5’ UTR, CDS and terminators | BpiI |
| Level 1 | Composite | Transcriptional units | BsaI |
| Level 2/M/P | Composite | Multiple transcriptional units | BpiI |
For synthesizing basic parts, the part of interest should be flanked by a BpiI site and its specific type overhang. These parts can then be cloned into the respective level 0 MoClo parts. For level 1, where individual transcriptional units are cloned, the overhangs come from the backbone you choose. The restriction sites for level 1 are BsaI. However, any type IIS restriction enzyme could be used.
Table 2: Type specific overhangs and backbones for MoClo. Green indicates the restriction enzyme recognition site. Blue indicates the specific overhangs for the basic parts
| Basic Part | Sequence 5' End | Sequence 3' End | Level 0 backbone |
|---|---|---|---|
| Promoter | NNNN GAAGAC NN GGAG | TACT NN GTCTTC NNNN | pICH41233 |
| 5’ UTR | NNNN GAAGAC NN TACT | AATG NN GTCTTC NNNN | pICH41246 |
| CDS | NNNN GAAGAC NN AATG | GCTT NN GTCTTC NNNN | pICH41308 |
| Terminator | NNNN GAAGAC NN GCTT | CGCT NN GTCTTC NNNN | pICH41276 |
References
- Lou, C., Stanton, B., Chen, Y.-J., Munsky, B., & Voigt, C. A. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology, 30(11), 1137–1142.
- Yang, S., Liu, Q., Zhang, Y., Du, G., Chen, J., & Kang, Z. (2017). Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology. ACS Synthetic Biology, 7(1), 287–291.
| None |