Measurement
The purpose of our measurement method was to determine the response function of the relay system to the input signal. The independent variable of the function is the intensity of the transcriptional output from the upstream module, which improve the functional predictability when combining the relay module with other biological modules like various micromolecule-induced promoters.
Therefore, based on the steady-state measurement framework from the previous three studies (Lou, Stanton et al. 2012; Zhang, Chen et al. 2016; Zong, Zhang et al. 2017), we designed our experiment and measurement scheme.
We first proposed a theoretically more accurate method:
According to the requisite condition of our model, we needed to cultivate the circuit-implanted cell in a balanced state for a time period, in which the binding of attB and attP sites reached equilibrium and achieved recombination.
We used the inducible promoter Ptet to construct the reporting plasmid—thus the reporter gene was not expressed during the integration and the recombinant reaction of recombinase with DNA—to avoid the possible growth burden caused by reporter gene expression, which lead to the deviation between actual proportion of the reorganized cells and the experimental statistics. The inducing process of the recombinase and the induced expression of reporter gene were also separated so that when the reporter gene was induced to express, the inducer as well as the intracellular recombinase concentration in the culture medium of the previous step were diluted instantly, and the recombinant reaction was ceased.
The detailed procedures are as follows:
M9 medium (supplemented）: 6.8 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 0.34 g/L thiamine, 0.2% casamino acids, 0.4% glucose, 2mM MgSO4, and 100 μM CaCl2
All incubations were carried out using a Digital Thermostatic Shaker maintained at 37 °C and 1000 rpm, using Corning flat-bottom 96-well plates sealed with sealing film. The cultivate medium contained an appropriate concentration of antibiotic.
1. Co-transfer the recombinase-expressing plasmid and cis-element-containing plasmid into the host E. coli DH5α.
2. Inoculate monoclonal colony in the M9 supplemented medium to avoid the strong leakage activity of promoter Ptac caused by the lactose residue in the LB medium.
3. Then the cell cultures were diluted 196-fold with M9 supplemented medium.
4. After 3 h of growth, the cultures were further diluted 700-fold with M9 supplemented medium of various IPTG inducer concentrations, and were incubated for another 6 h.
5. For the Ptac-sfGFP system that is regarded as the benchmark of the induced expression intensity, 15 μL samples of each culture were transferred to a new plate containing 135 μl per well of PBS supplemented with 2mg/μL kanamycin to terminate protein expression, and were kept in 4℃ condition for measurement.
6. Dilute the culture from step 4 200-fold again with M9 supplemented medium containing 50 ng/μL aTc inducer and cultivate overnight.
7. 3-μl samples of each culture were transferred to a new plate containing 200 μl per well of PBS supplemented with 2mg/μL kanamycin to terminate protein expression. The fluorescence distribution of each sample was assayed using a flow cytometer with appropriate voltage settings. The ratio of fluorescent cell was determined using FlowJo software .
We used the methods above to measure the IPTG-[sfGFP] (input) curve as well as the [input]-phiC31 recombination rate curve of the recombinant system. The results were fitted by modelling, as shown in blue in Figure 1:
Figure 1
However, this method is too complicated, and requires a considerable amount of materials. Thus, we expected to use a simpler measurement in our experiment.
In the simplified method, we applied the constitutive promoter instead of the inducive promoter the reporter gene originally possessed. We hoped to dilute the culture only for once and amplified cultivation before measuring. In this way, we anticipated that after the dilution, the cells could remain long-term logarithmic growth to satisfy the condition of equilibrium state. After entering the growth stable period, although the recombinase may further accumulate exceeding the precise measurement method mentioned above, the plasmids which underwent recombination would have a decreasing reporter gene expression along with the change of intracellular environment and eventually be distinguished from the cell population that recombined during the equilibrium period. We expected this method could yield at least consistent results of the response range with the precise method.
The detailed procedures are as follows:
1. Co-transfer the recombinase-expressing plasmid and cis-element-containing plasmid into the host E. coli DH5α.
2. Inoculate monoclonal colony in the M9 supplemented medium to avoid the strong leakage activity of promoter Ptac that is caused by the lactose residue in the LB medium.
3. The cell cultures were diluted 1000-fold with M9 supplemented medium of various IPTG inducer concentrations, and were incubated for 20h.
4. 3-μl samples of each culture were transferred to a new plate containing 200 μl per well of PBS supplemented with 2mg/μL kanamycin to terminate protein expression. The fluorescence distribution of each sample was assayed using a flow cytometer with appropriate voltage settings. The ratio of fluorescent cell and the arithmetical mean of each sample was determined using FlowJo software.
The [input]-phiC31 recombination rate curve of the recombinant system obtained from this method is shown in yellow in Figure 1. Though the fitting result is slightly different, the response ranges are basically identical, so that the key performance of the module can be described.
[1] Lou, C., et al., Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology, 2012. 30(11): p. 1137-42.
[2] Zhang, H.M., et al., Measurements of Gene Expression at Steady State Improve the Predictability of Part Assembly. ACS Synthetic Biology, 2016. 5(3): p. 269-273.
[3] Zong, Y., et al., Insulated transcriptional elements enable precise design of genetic circuits. Nat Commun, 2017. 8(1): p. 52
