Methodology in synthetic biology reflects basic principles in engineering, and the engineering cycle reigns amongst one of the most significant ones. The engineering cycle offers comprehensive and systematic guidance on the design and test of biological systems and consists of four stages: design, build, test, and learn.
We propose a programmable logic framework, LANTERN, capable of switching between different logic gates based on user requirements. We are fully aware that a standardized engineering cycle is even more critical to our cross-disciplinary work, and we followed it strictly and explored it innovatively.
We have reached decent results with the implementation of it, and a summary is as follows.
- We designed and refined the genetic circuits utilizing CRISPRi & recombinases.
- We conducted orthogonality verification to test the orthogonality of recombinases.
- We implemented directed evolution to improve the efficiency of recombinases.
Cycle 1: Genetic Circuit Completion
Design
Our initial circuit design incorporates four recombinases and CRISPRi to implement 16 possible logic gates (Figure 1).
In our design, CRISPRi acts as a crucial NOT gate. To verify its effect, we decided to simulate the repressive impact of CRISPRi on the target gene in Simbiology while proceeding with plasmid construction.
Build
Based on the mechanism of CRISPR interference, we establish reaction equations to demonstrate the inhibitory effect of dCas9 and sgRNA on the expression of GFP. We used Simbiology to solve ODE and simulate the production of GFP protein.
Test
We obtained the change curves of GFP in two conditions: with and without expression of dCas9 (Figure 2, 3). Our results confirmed that the combination of dCas9 and sgRNA effectively inhibits GFP expression, indicating that CRISPRi functions as anticipated in this context and implements the NOT gate function in our circuit.
However, during discussions among wet lab and model members regarding the application of CRISPRi in our genetic circuit, a challenge emerged: if the downstream promoter is inhibited by CRISPRi, normal expression of the gene of interest (GOI) cannot be achieved due to the steric hindrance caused by the dCas9 protein, even when the upstream promoter is actively initiating transcription.
Learn
While CRISPRi successfully implements the NOT gate function in our circuit, we need to solve the problem of its unexpected repression of upstream gene expression. So we added a patch (Figure 4) utilizing another two recombinases (Cre & fimE) to Registers, thus completing our genetic circuit design.
Cycle 2: Verification of recombinase efficiency and orthogonality
Design
Our project is based on site-specific recombination systems involving four different recombinases, making the orthogonality between these recombinases especially crucial. To verify this, we designed two types of plasmids: recombinase plasmids, each containing one of the four recombinases, and verification plasmids, each containing a pair of specific recombinase recognition sites. We transformed pairs of recombinase and verification plasmids into BL21(DE3) competent cells together and designed a pair of primers in the same direction, such that only the inverted verification plasmid could be detected through colony PCR.
Build
We successfully constructed the above eight plasmids by PCR and homologous recombination. Then, we transformed pairs of recombinase and verification plasmids into the BL21(DE3) competent cells for inducible expression using IPTG and verified our results using colony PCR.
Test
After induction with IPTG, we used colony PCR to verify the effect of recombinase inversing. The colony PCR results (Figure 6) show that these recombinases exhibit excellent orthogonality.
However, during the colony PCR, we found that some of the verification plasmids were still not inverted under the action of the corresponding recombinases, which indicates that the efficiency of the recombinases requires improvement.
Learn
From the results, the effect of these recombinases was not ideal. So, we decided to perform directed evolution using A118 as an example to improve the inverting efficiency of serine recombinases.
Cycle 3: Directed evolution of A118 recombinase
Design
An expression module and a selection module were designed. The expression module induces recombinase expression with IPTG, while the selection module uses a phage lysis gene φX174 E.
Build
We inserted the expression module into the pET-28a(+) vector and the selection module into the pET-22b(+) vector and transformed it into E. coli.
Test
We used epPCR to amplify the expression module to generate a random mutant library for the recombinases. After constructing the E.coli system, we conducted the screening plans to verify its functionality and recombination effect.
The inversion function of the recombinase was proved by the results of colony growth, indicating the reverse of φX174 E gene sequence and the suppression of the phage lysis gene.
The recombination efficiency was described by GFP fluorescence, which positively correlates to the expression level of the inverted GFP gene. Thus, the inversion efficiency of the recombinases can be characterized by the fluorescence intensity of GFP.
Learn
Though Traditional Mutagenesis allows for some mutations that have high efficiency, the process of constructing recombinase mutants and transferring them into E. coli is too tedious and labor-intensive. Therefore, we considered using a continuous directed evolution approach to reduce the workload.
Cycle 4: Continuous directed evolution
Design
Taking advantage of autonomous reproduction of bacteria, we can construct a persistent high-mutation system for continuous mutations during DNA replication. Therefore, we designed a bistable switch that allows reporter GFP inverted by the A118 recombinase under certain conditions and then inverted back by the Gp44 protein under different conditions, enabling a continuous selection process. To achieve continuous high mutation rates inside E. coli, we introduced eMutaT7, a fusion of cytosine deaminase (AID) and T7 RNA polymerase (T7RNAP).
Build
We designed three plasmids, pHyo094, Target plasmid, and Selection plasmid, to provide eMutaT7 enzyme, regulate A118 and Gp44 expression, and indicate the selection result of the continuous evolution. Each consists of different antibiotic resistance genes to facilitate co-transformation into E. coli.
Test
After transferring the three plasmids into E.coli, we induced the expression of eMutaT7 to mutate the A118 recombinase sequences. If it maintains its biological function, the 'Turnover' switch can execute to inverse the GFP gene, expressing protein and producing fluorescence. After sampling the strain, we removed the mutation inducers, activated the 'Turnback' switch, and turned the GFP gene back to its original state. After multiple cycles, we can screen for mutant recombinases.
Learn
It has been demonstrated that the efficiency of recombinases can be improved through directed evolution (Sclimenti et al., 2001). We plan to apply this approach to other suboptimal recombinases. We will conduct further verification at the total circuit level with these enhanced recombinases.
References
Sclimenti, C. R., Thyagarajan, B., & Calos, M. P. (2001). Directed evolution of a recombinase for improved genomic integration at a native human sequence. Nucleic acids research, 29(24), 5044–5051. https://doi.org/10.1093/nar/29.24.5044
