Difference between revisions of "Part:BBa K2918014"

Revision as of 21:30, 18 October 2019

Universal RBS

Ribosome Binding Site which is functional in both gram positive and gram negative bacteria.

Sequence and Features

Overview

This ribosomal binding site has been made by the use of the RBS calculator 2.0 by (Yang et al, 2017). This RBS functions both in gram negative (E. coli) and gram positive (B. subtilis). It has been demonstrated to work in P.putida .

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 [http://www.addgene.org/47992/ 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. The complete sequence of our parts including backbone can be found here.

Table 1: Overview of different level in MoClo

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

References

• Temme, K., Hill, R., Segall-Shapiro, T. H., Moser, F., & Voigt, C. A. (2012). Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic acids research, 40(17), 8773–8781.