Part:BBa_K5186002:Design
sRNA2(IspH)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
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.
Source
Synthetic oligo