Plasmid construction

The Register plasmid is constructed by ourselves. We used PCR to construct the designed primers into fragments of about 500bp. Agarose gel electrophoresis of some fragment extensions is shown below (Fig.1, and all others).

Figure 1: Agarose gel electrophoresis of extended PCR fragment

After successfully constructing these short fragments, we assembled them using overlap PCR and subsequently combined them with the backbone through homologous recombination. Same method is used to construct other plasmids.

Orthogonality of recombinases

Our Register part utilizes site-specific recombination systems involving four different recombinases, making the orthogonality between these recombinases a critical factor. 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. These plasmids were transformed by pair into E. coli BL21(DE3) competent cells (fig. 2a), and colony PCR was performed using a pair of primers designed in the same orientation (fig. 2b). This setup ensured that only inverted verification plasmids were detected. The results demonstrated excellent orthogonality between these recombinases (fig. 2c).

Figure 2: (a) Recombinase plasmids and verification plasmids transformed by pair (4 x 4). (b) Primers used in colony PCR. (c) Colony PCR results.

qPCR

We used qPCR to verify whether the expression of these recombinases can modify the Register plasmid, which consists of orthogonal recombinase recognition sites to evaluate their efficiency.

We inserted the Bxb1 recombinase gene in plasmid pET28a(+) and transformed the Bxb1-pET28a(+) plasmid with Register plasmid into E.coli BL21 (DE3) competent cells. Single colonies were selected, cultured, and induced with 1mM IPTG at 37° for four hours. We then extracted the plasmids from both induced and original cultures, and used the inverted and original plasmid samples for qPCR. We designed two pairs of qPCR primers(fig. 3) to respectively detect the original and modified Register plasmids to evaluate the inversion or not change. The result showed this process working properly (fig. 4).

Figure 3. Primers used in qPCR.

Figure 4. qPCR fluorescence intensity of Register A before and after being incubated by bxb1 recombinase.

As the initial template amount undergoes exponential amplification in relation to the Ct value, we can evaluate recombination rates by comparing the Ct values between different pairs of primer within the same sample. By comparing the Ct values of the inverted and original Register plasmids, we can assess the efficiency of these recombinases using the following formula:

We assumed that each one-cycle difference in Ct value corresponds to a doubling of the initial template amount. The qPCR fluorescence results (table.1 &fig. 5) underscore the exceptional performance of our Register system.

Figure 5. Recombinases efficiency calculate from Ct value. 'Undetermined' indicates that the Ct value exceeds the maximum number of amplification cycles (40) and is therefore calculated as 40.

Sequencing

We used Bxb1 as an example to verify whether recombinases function properly. Original and Bxb1-incubated Register A plasmids were both sequenced to obtain the precise sequences(fig. 6). The results confirmed that Bxb1 excise the target gene correctly.

Figure 6. Sequence of original and Bxb1 induced Register A

Register 0 induction

To verify whether two or more promoters can function collaboratively, we first construct Register 0 plasmid as proof-of-concept for Register part (fig. 7). Register 0 includes overlapping and orthogonal recombinase recognition sites, four promoters(constant promoter J23111, trc promoter, and inducible promoter lac operon, Rha promoter) with different directions.

Using Register 0, we tested two key aspects: first, whether two promoters in series could achieve the functionality of an OR gate; and second, whether the activity of a constitutive promoter in the reverse orientation would interfere with the expression driven by a forward-oriented promoter.

Figure 7: Design of Register 0

The distance between the promoter and the start codon should be minimized for optimal efficiency. Therefore, we placed the necessary elements of the inducible promoter such as LacI, rhaS and rhaR outside the recombinase sites, ensuring that only promoters are in the register region (fig. 8).

Figure 8: Register 0 plasmid

After the plasmid was successfully constructed, we transferred the Register 0 plasmid into BL21 (DE3) competent cells. Once they reached the logarithmic growth phase, induced the cells by adding 1mM IPTG and 0.3% rhamnose at a final concentration. We recorded the time span and fluorescence intensity as output.

The results shows that both IPTG and rhamnose (rha) induce expression effectively, and simultaneous induction of both promoters leads to enhanced expression (fig. 9). This indicates that the Register 0 system functions as an excellent OR gate. Since the distance between the Lac operon and the start codon is farther than that of the rhamnose promoter, the Lac operon has a smaller effect on mRNA transcription when the rhamnose promoter is active.

Figure 9. OR GATE test of Lac operon and rhamnose promoter.

Directed evolution

We use directed evolution to enhance recombinases efficiency. Initially, we employed traditional directed evolution strategies, including error-prone PCR and the eMutaT7 directed evolution system, but these approaches are proved to be inefficient. Consequently, we shifted our focus towards enhancing recombinase efficiency through rational design.

Since the structure of the recombinase-DNA complex is unresolved, we selected the A118 recombinase as an example and predicted its composite structure with the attP site using AlphaFold 3 (fig. 10). Molecular docking was then performed to study their interactions (fig. 11). Moving forward, we plan to conduct directed evolution on this region to enhance activity of A118 and identify key amino acid residues for NNK mutagenesis.

Figure 10. molecular animations of the predicted structure of A118 and attP.

Figure 11. A118-dsDNA complex and core residue identified.

Supplement

The following are the results of our colony PCR verification using different primers, after we transfected the recombinase plasmid and the verification plasmid together and induced them with IPTG; Each fragment on the gel image is marked with two letters and a number, where the first letter represents the recombinase corresponding to the recombinase plasmid, the second letter represents the recombination site corresponding to the verification plasmid, and the number represents the parallel repeated test. (Abbreviations of recombinases: A-A118, B-Bxb1, C-Cre, F-Fime, T-Tp901, φ-PhiC31)