Part:BBa_K3868097

pCBE

The pCBE plasmid contains the lambda operator, cytidine deaminase, Uracil DNA glycosylase inhibitor and LVA degradation labels. The CBE / sgRNA complex can bind to the double-stranded DNA to form an R-loop in a sgRNA and PAM-dependent manner. CDA catalyzes the deamination of cytosines located at the top (non-complementary) strand within 15–20 bases upstream from PAM, which results in C-to-T mutagenesis.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1572
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1572
  Illegal NheI site found at 1331
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1572
  Illegal BglII site found at 5013
  Illegal BamHI site found at 3610
  Illegal XhoI site found at 4616
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1572
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1572
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

        The dual plasmids system of CBE was designed, built and tested. The pCBE plasmid contains the lambda operator, cytidine deaminase, Uracil DNA glycosylase inhibitor and LVA degradation labels. Based on the pCas, the pCBE (BBa_K3868097) was successfully constructed (Fig. 1A). To extend the editing range, two different sgRNA expression frames were tandemly linked, allowing GGGGGGGG to be covered, resulting in a more diverse editing outcome. Based on the pTarget, the pTargetS plasmid (BBa_K3868098) was successfully constructed (Fig. 1B), and the sequences of sgRNA1 and sgRNA2 was showed in Fig. 1C. The CBE / sgRNA complex can bind to the double-stranded DNA to form an R-loop in a sgRNA and PAM-dependent manner. CDA catalyzes the deamination of cytosines located at the top (non-complementary) strand within 15–20 bases upstream from PAM, which results in C-to-T mutagenesis.

Fig 1. A and B. The dual plasmid system was designed and used for CBE system. C. A schematic model for CBE.

Results

         During construction of the library, 90 single colonies were randomly selected for sequencing. It was found that 48 variants with different RBS sequence were identified from 90 samples, with an editing efficiency of only 53%. However, it is noteworthy that the transformants were grown on the solid medium for longer time, the reproducibility of the results gradually increased, and majority of variant RBS sequences became GAAAAAAG (Fig.2), probably due to the continuous base editing in the transformants. The above results show that although CBE possesses the advantages of simplicity and rapidity, editing results and efficiency applied in BL21 (DE3) are not sufficiently stable.

Fig 2. Schematic representation of the changes in G and A abundance of the RBS variant sequences of T7 RNAP obtained from CBEs experiments.

Reference

1. Gong G, Zhang Y, Wang Z, Liu L, Shi S, Siewers V, Yuan Q, Nielsen J, Zhang X, Liu Z. GTR 2.0: GRNA-tRNA array and Cas9-ng based genome disruption and single-nucleotide conversion in Saccharomyces cerevisiae. ACS synthetic biology. 2021; 10: 1328–1337.

2. Zhao D, Li J, Li S, Xin X, Hu M, Price MA, Rosser SJ, Bi C, Zhang X. Glycosylase base editors enable C-to-A and C-to-G base changes. Nature Biotechnology. 2021; 39: 35–40.

Improve From NJXDF-CHN 2022

Group: iGEM22_NJXDF-CHN

Author: Yang Gu

Overview

         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.

Design

         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.

Fig. 1 The plasmid construction process

        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.

Fig. 2 (A) The gene after the special SD sequence cannot be produced properly at low-rate expression rate (B) The secondary structure of special SD sequences.

Result

        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.

Fig. 3 (A) The test workflow for CBE editorial moderation. (B) The editing efficiency under different induction times at 42℃.

        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.

Fig. 4 (A) The workflow of pET plasmid RBS library construction and screening. (B) The unit cell fluorescence intensity of the fermented culture Alloferon-1.

References

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.