Team:NAU-CHINA/RESULTS/improvement
IMPROVEMENT
Background
As an important part of mRNA, bacterial 5’ untranslated regions (5’ UTR) involves in a complex regulation of gene expression. However, the exact sequence features contributing to gene regulation are not yet fully understood. But many research 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. Therefore 5’ UTR 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(Fig.1). 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.
Fig.1 Dual UTR(BBa_K3735020)
Our Design
After learning about the principle of Dual UTR, we wanted to utilize it to improve an existing measurement part so that subsequent researchers can detect fluorescence more efficiently and accurately. We chose the BBa_J04450 which expresses mRFP under the induction of IPTG as our target sequence. We intended to add the Dual UTR between mRFP(BBa_E1010) and lac promoter(BBa_R0010) to improve RFP expression. Thereupon, we construct the following part(BBa_K3735666).
Fig.2 Improved mRFP expression element(BBa_K3735666)
Characterization:
We constructed the expression plasmid(K3735020-pSB1C3) which Dual UTR sequence was inserted by using homologous recombination. Then the recombinant plasmid was transformed into E.coli BL21 competent cells and grown to a 0.6 optical density at 600nm(OD600) . Single colonies were inoculated to 50ml LB medium containing chloramphenicol and cultured for 12 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.
Zhou L, et al. Construction and antitumor activity of selenium nanoparticles decorated with the polysaccharide extracted from Citrus limon (L.) Burm. f.(Rutaceae)[J]. International Journal of Biological Macromolecules, 2021, 188:904-913.
El-Saadony Mohamed T. et al. The use of biological selenium nanoparticles to suppress Triticum aestivum L. crown and root rot diseases induced by Fusarium species and improve yield under drought and heat stress[J]. Saudi Journal of Biological Sciences, 2021, 28(8):4461-4471
Zohra Efat et al. Potential applications of biogenic selenium nanoparticles in alleviating biotic and abiotic stresses in plants: A comprehensive insight on the mechanistic approach and future perspectives[J]. Green Processing and Synthesis, 2021, 10(1):456-475.
Azimi Fatemeh et al. Chitosan-selenium nanoparticles (Cs-Se NPs) modulate the photosynthesis parameters, antioxidant enzymes activities and essential oils in Dracocephalum moldavica L. under cadmium toxicity stress.[J]. Plant physiology and biochemistry : PPB, 2021, 167:257-268.
Yuting Wang. Screening of nano-selenium-producing strains and selenium-producing mechanism and application study [D]. University of Science and Technology of China, 2019.
Pengfei Lu et al., Effects of exogenous Selenium fertilizer on grain yield and Plant Selenium accumulation in wheat [J]. Journal of Agricultural University of Hebei, 2020, 43(03):17-22.