Part:BBa_K3735666

enhanced BBa_J04450

Bacterial 5’ untranslated regions as an important part of mRNA (5’ UTR), it involves in a complex regulation of gene expression; however, the exact sequence features contributing to gene regulation are not yet fully understood. But many researches have proved that 5’ UTR plays a central role in gene expression and protein production. At the DNA level, it involves in transcript formation for the interplay between promoter and initially transcribed sequences (ITS). But, at mRNA level, it influences transcript stability and translation because of secondary structure formation and so on. Therefor 5’ UTR is has a crucial contribution to the maintenance of a fine balance between transcription, transcript stability and translation. In the work of Simone Balzer Le, Ingerid Onsager et al, they design a novel 5’UTR-Dual UTR, utilizing the transcriptional and translational characteristics of 5’UTRs in a single expression cassette, which will improve protein expression greatly.

The Dual UTR contains two different UTRs which separated by a spacer region (Fig1). The 5’ UTR proximal to the promoter originates from the transcriptional screening(Tr-UTR) which affects transcript formation, while the second 5’ UTR is identified from the translational screening (Tn-UTR) which influences transcript stability and translation. And the spacer region provides enough space for physical separation of mutations affecting transcription and translation. By using the Dual UTR, we can improve the protein expression greatly.

Figure.1 Dual UTR(BBa_K3735020)

Our design: After learning about the Dual UTR, we want to utilize it to improve an existing measurement part so that subsequent researchers can detect fluorescence more efficiently and accurately. Then we chose the BBa_J04450 which expresses mRFP under the induction of IPTG. We intend to add the Dual UTR between mRFP(BBa_E1010) and lac promoter(BBa_R0010) to improve its expression. Thereupon, we construct the following part(BBa_K3735666).

Fig.2 Improved mRFP expression element(BBa_K3735666)

Characterization: We constructed the expression plasmid(BBa_K3735666) by using homologous recombination, which with Dual UTR sequence inserted. Then the recombinant plasmid was transformed into E.coli BL21(DE3) competent cells and grown overnight. Then the single colony(BBa_K3735666) and control(BBa_J04450) were used to inoculate in 50ml LB medium containing chloramphenicol and cultured to the same state. We cultured them under the induction of IPTG, and measured OD₆₀₀ and fluorescence intensity per 2 hours.

Fig.3 Plasmid map(A: Improved part (BBa_K3735666); B:Original part(BBa_J04450))

Results:

Fig.4 The results of improvement. A: the value of OD₆₀₀ of BBa_J04450 and BBa_K3735666; B: the fluorescence intensity of BBa_J04450 and BBa_K3735666; C: the fluorescence intensity/OD₆₀₀ of BBa_J04450 and BBa_K3735666

We can see that the fluorescence intensity of the improved part(BBa_K3735666) is significantly higher than that of the control group(BBa_J04450). Increasing the expression of fluorescence intensity can amplify weaker signals. At the same time, our method is also suitable for improving the expression intensity of other parts.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 882
  Illegal AgeI site found at 994
  
• 1000
  COMPATIBLE WITH RFC[1000]