Difference between revisions of "Part:BBa K4275030"

Revision as of 10:13, 12 October 2022

pRha-riboj

The part uses a rhamnose-inducible promoter system to achieve high level expression of GOI with the fusion of a ribozyme at the junction of the promoter and its downstream sequence.

The part is an improved version based on existing pRha part (BBa_K914003), which is characterized by its high protein expression ability. The part was later modified in the construction of pRha-riboJ-Neae-Nb to enhance expression of Nb which enables cellulosome complex to be displayed on surface of E.coli.

Usage and Biology

Ribozyme (ribonucleic acid enzymes) are RNA molecules with a variety of different functions including RNA splicing in gene expression. A synthetic self-cleaving ribozyme called RiboJ was used in this circumstance to increase the RFP expression.

RiboJ is a 75 nucleotide sequence consisted of a satellite RNA of tobacco ringspot virus derived ribozyme and a 23 nucleotide hairpin. During post-transcriptional processing of mRNA, RiboJ self-cleaves to eliminate its upstream sequence and remove the promoter-associated RNA leader. Consequently, only the uncleaved hairpin sequence is presented before the RBS and gene of interest. This genetic insulation would increase gene expression level at both RNA and protein level. Gene insulation by RiboJ increases gene expression via stabilizing mRNA and reducing the degradation of the transcript, resulting in higher transcript abundance. The greater mRNA abundance is then amplified by translation, elevating the production of target protein.

Sequence and Features

References

1.Clifton, Kalen P et al. “The genetic insulator RiboJ increases expression of insulated genes.” Journal of biological engineering vol. 12 23. 29 Oct. 2018, doi:10.1186/s13036-018-0115-6