Part:BBa_K2047101

Fluorescent reporter system with stem-loop of -30.1kcal/mol, GFP_stem-loop

Introduction

Inspired by Xu’s work and based on keasling’s work, we designed a series of stem-loops with different free energy for further use as basic regulatory parts. To measure the regulation effect of stem-loop, we constructed the dual-fluorescent reporter system (GFP and mCherry) to test the regulatory effect of various stem-loops.

The operon is transcribed by its sole promoter and the primary transcript is cleaved into several secondary transcripts by RNase E, a single-stranded, nonspecific endonuclease with preference for cleaving A/U-rich sequence. However, the stability of these secondary transcripts against exonuclease degradation from the 3’ end varied due to their distinct terminal structure. When stem-loops inserted in the 3'end of the upstream gene, it protects its mRNA against the cleavage of exonuclease, increasing the ratio of abundance of the first gene product relative to that of the second gene product. Furthermore, the lower free energy of stem-loops are, the more stable the secondary transcripts of the upstream are, tuning the expression of multiple genes.

Figure 1 shows the expression of fluorescence protein effected by stem-loops with different folding free energy. It’s clearly that the difference is significant. Then we measured the quantitative expression on two levels: the transcriptional level and the translational level.

Figure.2 shows the relative expression level of the dual-fluorescence reporter system we designed. The ratio of GFP and mCherry has significant difference both on the transcriptional level and the translational level with different stem-loops.

Description

For the convenience of using and measuring the effect of the stem-loop, we also submitted a serious of parts which encode GFP with stem-loop and a RNase site.

This part encodes GFP with stem-loop of -30.1 kcal/mol that designed by ourselves and a RNase site downstream. The effect of transcript protection we measured of the stem-loop and RNase is as follows:

Figure 3 Shows the structure of the stem-loop of this part with the folding free energy of -30.1 kcal/mol.

Figure 4 Shows the relative expression on RNA and protein level with stem-loop of -30.1 kcal/mol (measured by Mfold) contrast to the control group with no stem-loop. The result is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.(***P value<0.0001, *P value<0.05)

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 777
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 644
  Illegal BsaI.rc site found at 768