Part:BBa_K5186002

sRNA2(IspH)

Description

sRNA(IspH)s are synthetic small regulatory RNAs (sRNAs) designed to specifically down-regulate the expression of the IspH gene in E. coli, thereby enhancing HMBPP production for mosquito attraction. Each sRNA(IspH) is composed of a target-binding sequence that is complementary to the IspH mRNA, enabling hybridization, and a SgrS scaffold that facilitates the recruitment of the Hfq protein in E. coli. This interaction promotes the sRNAs' binding to ispH mRNA and expedites its degradation by RNase E. (Seung Min Yoo et al.)

It has been revealed that the mutation in the SgrS scaffold can enhance the efficiency of sRNA-mediated gene repression (Minho Noh et al.). This year, we have successfully engineered 4 variants of sRNA(IspH)s in E. coli, each with a specialized scaffold. We expect to select the best one for controlling the down-regulation of IspH from them. And sRNA2(IspH) introduces a 6-nts stem with the SgrS-S scaffold, exhibiting the most excellent downregulation effect without affecting bacterial growth. These sRNA(IspH)s (BBa_K5186001, BBa_K5186002, BBa_K5186003, BBa_K5186004, BBa_K5186005) for the downregulation of downstream gene expression and various MEP overexpression cassettes (BBa_K5186006, BBa_K5186007, BBa_K5186008, BBa_K5186009) contribute to the overproduction of HMBPP in E. coli and thus make up a part collection. This collection can help and inspire other iGEM teams and researchers to achieve higher yield of HMBPP or other isoprenoids in E. coli.

Usage and Biology

In E. coli, IspH is an essential enzyme in the MEP pathway, crucial to isoprenoid synthesis which helps maintain, stabilize and support core functions such as respiration. It catalyzes the conversion of HMBPP into the common isoprenoid precursors IPP and DMAPP in a single step. And it has been confirmed that significantly inhibiting the IspH activity in E. coli substantially resctricts the bacterial growth (Kumar et al.). To achieve a balance between HMBPP overproduction and growth activity of E. coli, we decided on the sRNA-based down-regulation of IspH instead of the knockout approach.

The design of sRNAs is mainly inspired by trans-acting Hfq-dependent sRNAs, which binds at or near the ribosome-binding sites(RBSs) of target mRNAs with the aid of the RNA charperone Hfq. This binding prevents ribosomes from accessing the RBS, thereby inhibiting translation. (Seung Min Yoo et al.)

In our study, sRNA2(IspH) is inducibly expressed under the control of pTac (BBa_K5186005) while overexpressing DXS, IspG and IspDF. Upon expression, sRNA2(IspH) binds to Hfq protein, aligning its target-binding sequence with the IspH mRNA. This interaction prevents ribosomes access to the RBS, effectively downregulating IspH expression and allowing for precise control over HMBPP synthesis without decreasing E. coli's viability.

Characterization

Figure 1.4 variants of sRNA(IspH)s are engineered in E. coli for down-regulation of IspH. (a) A graphical abstract of the molecular mechanism underlying the down-regulation of IspH expression by sRNA(IspH)s. (b) Genetic circuit and nucleotide sequences of sRNA(IspH)s expression. The green and blue sequences indicate the target-binding sequences and SgrS-S scaffold variants respectively. (c) Gel electrophoresis analysis of transformed sRNA(IspH)s expression cassettes. (d) Growth curve of control (E. coli strain DH5a) and strains expressing sRNA(IspH)s.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]