Part:BBa_K3805542:Experience
1. Construction of pathways
We synthesize PUC57 plasmids with proinsulin, proinsulin mutant 1, proinsulin mutant 2 genes, and obtain the araC gene by PCR from the plasmid given to us by Sichuan University, then obtain the GFP gene by PCR from the plasmid from our lab. After double digestion with the corresponding enzymes, we use T4 ligase to ligate these gene fragments to the PUC57 plasmids.
2. Validation of imported gene pathways
To verify successful vector construction, we introduce the constructed plasmids into trans 5αcompetent cells, plate and culture overnight. A single colony is picked for colony PCR to determine that both araC and GFP genes have been correctly inserted into the PUC57 plasmids. Finally, we store the engineered bacteria in glycerol for cryopreservation at -80℃.
3. mRNA switch feasibility experiment
(1) Bacteria internal experiments We use the E. coli BL21(DE3) strain containing a 2-pathway as the control group, and the E. coli BL21(DE3) strain containing proinsulin normal strain, proinsulin mutant 1 E. coli BL21(DE3) strain and proinsulin mutant 2 E. coli BL21(DE3) strain as the experimental group. Each strain is cultured at 200 rpm at 37 ℃ in 5 mL of medium, and the fluorescence is detected under the fluorescence microscope every 1 hour.
(2) Endocytosis experiments Proinsulin normal E. coli BL21(DE3) strain, proinsulin mutant 1 E. coli BL21(DE3) strain and proinsulin mutant 2 E. coli BL21(DE3) strain are experimental strains. We add 0μL,3μL,5μL,10μL 10μM/L 12bp mRNA fragments complementary to those at the 2-pathway mRNA switch for each 200μL bacterium. The bacterium are cultured at 200rpm at 37 ℃, and the OD600 and fluorescence intensity of the broth are measured every 30 min.
(3) mRNA turnover experiments In the experiment of RNA natural absorption, we obtain the relevant data that 12bp, 24bp and 36bp length RNA molecules all have an inhibitory effect on fluorescence production. We design RNA systems that inhibit the expression of the downstream kill system of the pathway. Data analysis shows that 12bp RNA is the most inhibitive. In order to exclude the effect of different absorption efficiencies caused by RNA length on the inhibition effect, we design an RNA turnover experiment. The experiment is as follows. Build engineered bacteria. The treatment of E. coli Trans 5αcontaining 1 and 2 pathways with a low temperature pre-cooled CaCl2 solution changed the permeability of its cell membrane and make it a competent cell that could easily absorb exogenous nucleic acid molecules. Heat shock transformation. We choose two RNA molecules of different lengths (12bp and 24bp in length, respectively) and set a concentration gradient of 0.005 smol/L, 0.01 smol/L, 0.03 smol/μL and 0.05 smol/L for each RNA for heat shock transformation. First of all, the receptor cells are frozen and thawed on the ice, in sterile conditions in accordance with the set concentration gradient to add RNA, gently mixed, incubated on ice bath for 30 minutes, and then incubated in a 42 ℃ metal bath for 90 seconds of static heat. Then centrifuge tubes are rapidly transferred to the ice bath and incubated for 5 minutes. 100 μL of bacteria solution is taken and added into 600 μL of LB liquid medium and cultured at 37 ℃, 200 rpm. Data processing and analysis. After transformation, we use microplate reader to measure OD600 and GFP's expression levels every 5 minutes. After a 60 minutes measurement, we turn to 30 minute measurements, collect the data and model the analysis.
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