Part:BBa_K3805542:Experience
mRNA switch
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.
Bacteria internal experiments
As we expected, the Normal E.coli should not glow fluorescent because the certain 12bp sites in proinsulin combined with complementary sequences in path 2. However, in the case for MutationⅠand MutationⅡ E.coli, it should glow flurescent as for sequences mutation.
But according to our results of bacteria internal experiments, the Normal, MutationⅠand MutationⅡ E.coli all could glow bright fluorescent within 1-12 hours, which means unfortunately mRNA switch had not work.
We proposed two possible reasons for the outcome after discussing with professors and analyzing the results. (1)12bp mRNA bounding site is not enough for mRNAs’ interaction. (2)The efficacy of this switch is poorly guaranteed. We continued to design a series of experiments to test it.
Endocytosis experiments
The results showed that if 12bp mRNA were added to the medium at the concentrations of 0.005μM/μL, 0.01μM/μL and 0.03μM/μL, the fluorescence intensity of bacteria containing the Pathway I + Pathway II system would become increasingly lower with the incubation time, indicating that bacteria could absorb short mRNA under normal conditions and the mRNA interaction could inhibit the expression level of green fluorescent protein. We also found that each generation of bacteria could continuously absorb it to maintain an overall continuous decline in GFP/OD600.
mRNA transformation experiments
We used mRNA transformation assay to artificially transform mRNA into cells and detect fluorescence intensity during the recovery period and the breeding period. As expected, bacteria can emit fluorescence, and its intensity decreases with time. The results showed that the fluorescence intensity per unit volume would decrease in 0-150 minute of the 12bp and 24bp mRNA transformation experiments. This proved that the simple 12bp and 24bp mRNA sequences could bind to certain sites in the mRNA transcribed from Pathway II and serve as switches. It meant that our vision of mRNA switching was possible to some extent.
However, we also found that the efficacy of the mRNA switch did not improve with the increase of mRNA concentration. The possible reasons may lay in the low transformation efficiency or the length of the mRNA binding sites. We will design further experiments to test the optimal length of mRNA binding site in the case of strain self-expression to truly reflect the efficiency of our switch.
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