Part:BBa_K4237022

PvanR_THS

Improve van induced promoter. RNA appratus is added downstream PvanCC

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 90
  Illegal NheI site found at 237
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 66
  Illegal BamHI site found at 104
  Illegal BamHI site found at 213
  Illegal BamHI site found at 251
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

This a an improvement part of Part:BBa_K3317006

THS strategy

The THS regulator encodes a structural component followed by a ribosome binding site (RBS) that is used to drive translation of the GOI. The structural region is designed to form a strong hairpin loop that when transcribed hinders the ability for ribosomes to bind the RBS, and thus inhibits translation. Translation is activated by expression of a complementary small RNA (sRNA) trigger that hybridizes to a short unstructured region of the THS, causing a breakdown in its secondary structure. This conformational change allows ribosomes to bind the RBS and translation of the GOI to proceed. THSs were selected because they offer strong repression of translation, can be designed computationally, and large libraries of designs exist with minimal crosstalk when used together

Figure. 3 Stragety of THS and performance comparison of single- and multi-level controllers in vivo. A. Stragety of THS B. Total GFP fluorescence for ‘off’ and ‘on’ input states (0 and 1 mM IPTG, respectively). Points show the three biological replicates for each controller and condition (black circles, Ptac; blue squares, THS; red diamonds, STAR; orange crosses, DC). Black dashed line denotes the mean fluorescence of cell autofluorescence (a.f.) controls containing no plasmid with grey shaded region showing ±1 standard deviation of 11 biological replicates. Fluorescence given in calibrated molecules of equivalent fluorescein (MEFL) units.

We tested the response of the food additive vanillic acid response promoter PvanCC and found that its activation ploidy was low compared to the original literature, and to further increase its activation ploidy, we used the literature-reported, RNA-coordinated THS strategy to increase the activation ploidy from approximately 20-fold to 34-fold.

Figure. 7 Characterization of PVan_CC and PVan_CC_THS.