Improving Part

    We reviewed the relevant work of NNU-China in 2021. This project utilized the cytosine base editor (CBE) and CRISPR/Cas9 technology to construct a ribosome binding site (RBS) library of T7 RNA polymerase (RNAP), which was applied for rapid screening of expression hosts suitable for antimicrobial peptide (AMP) production. However, we found that not all of the AMPs involved in the optimization had significant increases in yield. We hypothesized that the RBS sequence of the target gene in the pET plasmid also plays a decisive role in the recombinant protein yield. Therefore, we selected the relevant components used by the team, CBE (BBa_K3868097) and Alloferon-1 (BBa_K3868102), for enhancement. This enhancement is expected to be extended to the optimization of various pET series plasmids.

    To prevent over-editing, we replace the promoter in BBa_K3868097 with a temperature-sensitive promoter (obtained by BBa_Q04510), which can be used to manipulate the start and end of editing by temperature switching (PL-CBE) (Fig 1). Notably, the subsequent recombinant plasmids were constructed on the basis of the original plasmid pSC101 (CmR) or pET-24a (KanR) using the restriction-ligation method. Further, the recombinant plasmids were all transformed by electro-transformation. In brief, Escherichia coli BL21 (DE3) or derived strains were cultured in 4 mL LB medium (OD600 reached at 0.6-0.8). The above bacterial liquid was centrifuged at 4,000 rpm for 10 minutes at 4°C. Next, add 1 mL glycerol (10%), pipet up and down slightly to mix thoroughly. And centrifuge at 4,000rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard. The above steps need to be repeated once. Add 100 μL glycerol (10%) and recombinant plasmid or linearized fragment (100 ng or more) to the bacteria, and transfer to the electro-rotor cup. After electroshock (1.85kV, 200Ω, 25μF) using the MicroPulser electroporator, spread it on selected plates after 1h at 37℃. To test whether the editing process was controlled, we selected E. coli endogenous lacZ as a reporter gene (5’-caacagttgcgcagcctgaa-3’) for testing. The dilution of the bacterial solution under different conditions was spread on plates, containing resistant (Cm+Spec) and X-gal. The number of blue spots and white spots can be observed to determine the status of base editing.

    Further, to obtain the RBS library of pET plasmid rapidly, we replaced the RBS of pET-Alloferon-1-eGFP (BBa_K3868102) by referring to the NNU-China team (5'-CCGCCGGATTTACTAACTGGGGGGGGCACTAA-3'). The purpose of this step is to facilitate base editing of RBS using CBE. To enable convenient screening of optimal mutants, we refer to the high-throughput screening approach developed by Rennig et al. Specifically, we added a resistance gene (AmpR) with a stable secondary structure of SD sequence after the eGFP gene (Fig 2). When the preceding protein is expressed at a weaker intensity, the later-linked resistance genes cannot be translated properly, which plays a positive screening role. We incubated strains containing plasmids PL-CBE and RBS8G-sgRNA (BBa_K3868050) into 5mL of LB medium for 12-16h at 37°C. Then, we transferred the bacterial solution at a concentration of OD600=0.2 in 10 mL LB medium with inducer (IPTG) and associated antibiotics (Kan+Spec+Cm+Amp). We dilute and spread the bacterial solution under high concentration conditions, and the colonies were selected to 96 deep-well plates for culture. The above bacterial solution was aspirated 200μL into a 96-well plate and the unit fluorescence intensity was measured by microplate reader.

    Based on the improvement of the two components, we rapidly constructed an RBS library of the pET plasmid, leading to a further 5.4-fold increase in Alloferon-1 yield.

    It has been shown that extending the incubation time of the strain will increase the editing efficiency, which often leads to over-editing. To overcome this problem, we replaced the constitutive promoter PCas in BBa_K3868097 with the inducible promoter Plambda cI. Further, we transformed optimized CBE and sgRNA (targeting the lacZ gene) into BL21(DE3) for base editing test, which were induced at 42°C for 0h,6h, 12h, 18h, and 24h, respectively. The experimental results showed that the number of white spots was found to be positively correlated with the induction time (Fig 3A). After calculations, the editing efficiency was 8.6% (3/35), 30.7% (23/75), 78.2% (43/55), 86.7% (60/69), and 96.0% (69/72), respectively (Fig 3B). To ensure the authenticity of the experiment, we selected five white spots in each case for sequencing, and all were found to have introduced nonsense mutations in lacZ.

    In constructing the RBS library and high-throughput screening of pET plasmids, we drew on the NNU-CHINA team and the relevant contents of the literature. We ligated a resistance gene (AmpR) containing a specific SD sequence after the eGFP gene of BBa_K3868102, which can only be produced when the recombinant protein is expressed at high speed. With increasing concentrations of ampicillin, Also, we replaced the RBS sequence of the target gene in the pET plasmid to facilitate the construction of the RBS library using CBE. To facilitate the use of the system for future teams, we constructed the new composite part BBa_K4297077 (registered in 2022) by integrating the PL-CBE into the optimized pET plasmid. The obtained pET plasmid library was successfully cultured under high concentration of ampicillin (4000 μg/mL) in accordance with the steps in the design section (Fig 4A). We selected 96 different single colonies for culture and selected three strains with the highest unit fluorescence intensity for further fermentation. The experimental results showed that the unit fluorescence values of the three strains were further improved compared to the original study (up to 5.4-fold) (Fig 4B). Excitingly, the entire screening process takes only 5-7 days and has the potential to be used in conjunction with the RBS library of T7 RNAP.

    Rennig, M., Martinez, V., Mirzadeh, K., Dunas, F., Rojsater, B., Daley, D. O., & Nørholm, M. H. (2018). TARSyn: tunable antibiotic resistance devices enabling bacterial synthetic evolution and protein production. ACS Synthetic Biology, 7(2), 432-442.