Present Experiment
Worker Module
araC and pBAD promoter expression
1. Construction of pathwaysWe obtain the gene fragments of araC and pBAD promoter by PCR from the plasmid given to us by Sichuan University, and insert the required enzyme digestion sites at both ends. After the completion of PCR, electrophoresis is performed to determine the size of DNA, and then DNA fragments are recovered. Finally, we use the restriction endonucleases for digestion, and then ligate the araC and pBAD promoter fragments into PUC19 plasmids using T4 ligase. Subsequently, in order to verify the inhibitory effect of araC on pBAD promoter, we insert GFP (green fluorescent protein) downstream of pBAD promoter using the same method mentioned above. The intensity of GFP fluoresence represents the transcriptional level of pBAD promoter.
Figure 1 proinsulin normal 1+3-pathways
Figure 2 proinsulin mutant 1 1+3-pathways
Figure 3 proinsulin mutant 2 1+3-pathways
We transform E. coli BL21(DE3) competent cells with the prepared plasmid for overnight culture. Then, we confirm that the constructed plasmid has been successfully transformed E. coli Trans 5α by colony PCR as well as PCR using the extracted plasmid as the template. Finally, we preserve the engineered bacteria in glycerol and store them at -80℃ for standby.
3. Verification of the inhibitory effect of AraC on pBAD promoter(1) Inhibitory effect of araC on pBAD promoter
We select E. coli BL21(DE3) strain control, proinsulin normal E. coli BL21(DE3) strain, proinsulin mutant 1 E. coli BL21(DE3) strain and proinsulin mutant 2 E. coli BL21(DE3) strain as the four groups of experiment strains. We add 0.6μL 500mM/mL IPTG for each 200μL bacterium to induce the expression of proinsulin and araC fusion protein downstream of the lac operon, then incubate the bacterium at 37℃,200rpm, and the OD600 value and fluorescence intensity of the broth are measured every 30 min.
(2) Fusion expression of araC and proinsulin
We add 60μL 500mM/L IPTG for each 60 mL proinsulin normal strain, proinsulin mutant 1 E. coli BL21(DE3) strain and proinsulin mutant 2 E. coli BL21(DE3) strain to induce the expression of proinsulin and araC fusion protein downstream of the lac operon for 4h. The three fusion expression proteins are extracted using his-tag protein extraction kit and verified by SDS-PAGE gel.
mRNA switch
1. Construction of pathwaysWe 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.
Figure 4 proinsulin normal 1+2-pathways
Figure 5 proinsulin mutant 1 1+2-pathways
Figure 6 proinsulin mutant 2 1+2-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. Feasibility experiments on mRNA switch(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.
Guard Module
Plasmid Construction
The Guard's gene pathway is mainly composed of Toggle Switch and T7-agrC-agrA pathway. Toggle Switch includes Ptrc-Protein pathway and P2-LacI(LVA)-mCherry pathway. Originally, Ptrc-2 promoter is followed by the gene of target protein. However, to observe the expression of the target protein, we substitute the target protein with GFP, so as to monitor the expression level of the target gene easily.
Figure 7 Guard Plasmid
OmpT Function Verification
OmpT signal peptide is an excretion tag linked on the N-terminus of protein.We fuse OmpT signal peptide at the N-terminus of mCherry to transport mCherry across outer membrane. To verify the function of OmpT signal peptide, we place OmpT-mCherry on plasmid pETDuet-1 ,then transform the recombinant plasmid into E.coli DH5α as the test group. We also construct a recombinant pETDuet-1-mCherry plasmid without OmpT signal peptide and also transform it into E.coli DH5α as the control group. We use fluorescent microscope and microplate reader to evaluate the transportation efficiency of OmpT signal peptide.
We use the fluorescent microscope to take pictures of the mCherry. We carry out more accurate experiments with microplate reader and carefully analyze the mCherry fluorescent intensity data of the test group and control group. If mCherry can be secreted out of cells with OmpT signal peptide, the fluorescent intensity of the bacteria solution’s supernate after supernation will be distinctly higher than the precipitation compared to the control group. Therefore, we choose the difference of the mCherry fluorescent intensity between the supernate and precipitation as the index of transportation efficiency. The detailed description of this experiment is in the section “ OmpT Function Verification Protocols”.
Promoter Ptrc Examination
To examine the inhibition effect of LacI on promoter Ptrc, we insert GFP gene to the plasmid ptr99a, which contains lacI gene and then is linearized. The linearized product doesn’t contain lacI gene anymore and has homologous hangs on its ends for further self-circulation by in-Fusion cloning. We then obtain two plasmids (one with lacI, the other without) with GFP situated at the downstream of the promoter Ptrc. We then transform E.coli DH5α with this two kinds of plasmids respectively and measure the intensity of green fluorescence using fluorescent microscope and microplate reader to verify the inhibition efficiency of lacI on promoter Ptrc.
Toggle Switch Verification
To verify the efficacy of our Toggle Switch, we replace our target protein gene with GFP sequence. When a certain amount of exogenous AIP is applied to the bacterial fluid, red fluorescence(mCherry) is observed under the microscope. We then apply a concentration gradient of synthetic AIP to the bacterial fluid to establish an accurate coordination between AIP concentration and mCherry secretion and eventually plot the corresponding curve.
Killing Pathways and Parts Vertification
When the guard feels AIP, it will secrete mCherry to the outside of the cell, which can activate the cheater's own killing pathway. In this part of the experiment, we aim to verify mCherry's killing operation on cheaters.
Obtain fragments
The parts agrB and agrD are obtained from the genome of Staphylococcus aureus ; The pmrC and pmrA are obtained from the plasmid provided by NEU-China; The ccdB and GFP are obtained from the plasmid of our lab.
Construct the plasmids
In order to facilitate verification, we construct two plasmids, one is the original design with ccdB(plasmid-B), and the other uses GFP instead of ccdB(plasmid-G).
The obtained fragments are linked to the synthetic anti-mcherry pmrB. Finally, all the fragments are connected to pETDuet-1 plasmid and transformed into E. coli DH5α
Verify the correctness of the plasmids
The PCR products of colony are verified by gel electrophoresis to determine whether the length of the stripsare is correct. The plasmids are extracted and verified by enzyme digestion. Then the fragments are sequenced. The plasmid is transformed into E. coli BL21(DE3)(50ul).
Conduct confirmatory experiments
Expand the culture of bacteria containing plasmid-G, and then divide evenly into several parts. Add mCherry solution to one of them, and make loading tablets and observe the fluorescence under the fluorescence microscope. Then set the concentration gradient of mCherry solution, and measure the fluorescence intensity of GFP produced by bacteria with different mCherry fluorescence intensity by microplate reader.
Expand the culture of bacteria containing plasmid-B, and then divide evenly into several parts. According to the data measured in the previous experiment, mCherry solution with appropriate concentration is added to several of them and incubated for a period of time. The other is added with buffer as control. Apply the bacteria solution to the solid medium to form single colony. Count the number of growing colonies and calculate the mortality, so as to obtain the killing efficiency.
Cheater-guard Interaction Vertification
We culture a certain amount of cheaters for a period of time, and then put a certain amount of cheaters’ bacteria solution into the guards’ bacteria solution. Finally, we measure the amount of mCherry produced by the guard with an microplate reader.
Environmental Capacity and Glucose Consumption Measurement
Measure the environmental capacity
The same amount of three bacteria is inoculated in a certain amount of LB liquid medium. Use microplate reader to measure the value of OD600 every 30 minutes. Stop the detection when stability is reached. By modeling, we obtain the value of environmental capacity.
Measure glucose consumption
The amount of three kinds of bacteria’s environmental capacity is inoculated into SOC medium, and samples are taken every 5 minutes. After using theD-Glucose Content Assay Kit, the value of OD505 reaction is carried out by microplate reader, and then the absorbance is measured to obtain the concentration of glucose in the sample.
Further Experiment
Expression Burden Control Circuit
Obtain fragments
We synthesize the parts we need: PhtpG1,sgRNA(T7),sgRNA(PBAD),Pconst,dCas9.
Figure 11 Gene pathway map of Burden Control Circuit
Construct the plasmid
In order to facilitate verification, we construct a new plasmid to accommodate our new parts. Besides, we design a new plasmid only with T7 promoter, promoter and GFP. Finally, all the fragments are connected to pETDuet-1 plasmid and transformed into E. coli BL21(DE3).
Verify the correctness of the plasmids
The PCR products of colony are verified by gel electrophoresis to determine whether the length of the strip is correct.The plasmids are extracted and verified by enzyme digestion. Then the fragments are sequenced.
Conduct confirmatory experiments
A certain number of the bacteria is inoculated in a certain amount of LB medium. Use microplate reader to measure the fluorescence intensity of GFP produced by bacteria every 30 minutes.
Optimal Guard Ratio Vertification
According to the best guard ratio predicted by the modeling, a mixed experiment of three roles are designed to obtain the expected effect that the production efficiency is improved by using our platform through extracting the target protein. Then we fit with the modeling results to further optimize the hardware design.
