Plasmid construction

Since the Register plasmid contains many recognition sequences of recombinases and many repeated sequences, it is difficult and time-consuming to synthesize it by the company, so we chose to build it ourselves. We first used PCR to construct the designed primers into fragments of about 500bp. In each PCR reaction, we use the primers with complementary pairing at the 3' end and extension at the 5' end to extend the target fragment. Agarose gel electrophoresis of some fragment extensions is shown below.

Figure 1: Agarose gel electrophoresis of extended PCR fragment

After successfully constructing these short fragments, we connected them together using overlap PCR and finally combined them with the backbone using homologous recombination. We also used the same method to construct other plasmids.

Orthogonality of recombinase

Our project is based on the site-specific recombination system of six recombinases, so the orthogonality between different recombinases is particularly important. We designed twelve plasmids, divided them into six 'recombinase plasmids' and six 'verification plasmids,' and double-transformed them into bl21(DE3). Finally, we used colony PCR to verify our results.

Figure 2: Recombinase Plasmid

The six "recombinase plasmids" comprise six different recombinases and Plac promoters, which express the recombinases we need in the presence of inducer IPTG. At both ends of the recombinase gene of these plasmids, we designed a pair of primers (PET_28_T7_term and PET_28_T7, see Figure 2) for colony PCR to verify whether the recombinase plasmids had been successfully transferred.

Figure 3: Verification Plasmid

These six "verification plasmids" comprise recombination sites with opposite directions for six recombinases. Theoretically, if a recombinase matches the site, the DNA sequence between the recombination sites will invert. We designed a pair of primers (Test_F1_sk and Test_R1_sk) upstream and downstream of the recombination site and designed a reverse primer (Test_R_sk, see Figure 3) on the DNA sequence between the recombination sites. We first used Test_F1_sk and Test_R1_sk to check whether the verification plasmid was successfully transferred. Then, we used Test_F1_sk & Test_R_sk, and Test_R_sk & Test_R1_sk for colony PCR. If the sequence is not inverted, the group of Test_F1_sk & Test_R_sk will have obvious fragments; if the sequence is inverted, the group of Test_R_sk & Test_R1_sk will have obvious fragments.

The following are the results of our colony PCR verification using different primers, after we double-transfected the recombinase plasmid and the verification plasmid 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)

According to the gel image results, except for the Cre-recombinase plasmid, all other recombinase plasmids and verification plasmids were correctly transferred. The group double-transformed with the Cre-recombinase plasmid had no fragments, which requires our subsequent further verification.

After we confirmed that the plasmids had been successfully transferred, we used the two pairs of primers Test_F1_sk & Test_R_sk and Test_R_sk & Test_R1_sk for colony PCR to check whether our "verification plasmid" was inverted. The results are below.

Figure 6
The white marked fragments are the results of PCR using Test_F1_sk and Test_R_sk.
The red coded fragments are the results of PCR using Test_R_sk and Test_R1_sk.

Theoretically, if the "verification plasmid" has not inverted, the length of the white-marked fragments should be 587bp, and there should be no fragment marked in red-marked groups; if the "verification plasmid" has inverted, there should be no fragment marked in white-marked groups, and the length of the white-marked fragments should be 968bp.

The gel image shows that the orthogonality between the binding sites of different recombinases is good. Specifically, for the non-AA BB CC FF TT φφ group, the white-marked fragments except Aφ2 have clear fragments of the correct length; the red-marked ones have no clear fragments, and even if there are fragments, the length is not 968bp. Only AB2 FB2 Aφ2 Fφ2 TF2 have possible non-orthogonal recombinase site interactions. However, since these fragments only appear in one of the two parallel repeats, we suspect this may be related to the impurity of the plasmid we used.

For the AABBCCFFTTφφ group, the groups marked in white all had fragments of varying shades; among the groups marked in red, only the AA BB TT φφ group had a 968bp long fragment, and the color of the φφ group was lighter than AA BB φφ group. The fragments of white and red groups show that the four integrases A118 Bxb1 TP901 PhiC31 can all function, but their inversion effect is not very good, and there are some uninverted fragments. Therefore, we decided to improve the inversion efficiency of the recombinase through directed evolution. As for Cre and FimE, they do not belong to the serine recombinase family. Their failure to function correctly may be due to unsuitable strains or lack of auxiliary factors, which requires further experimental verification.

Register 0 induction

In order to verify whether two or more promoters can work together, in our Register parts, we first construct Register 0 plasmid as an example, which consists of some overlapping and orthogonal recombinase recognition sites, four promoters(constant promoter J23111, trc promoter, and inducible promoter lac operon, Rha promoter) with different directions. We used Register 0 to verify whether two promoters in a series can achieve the function of AND gate and whether the constant promoter in the opposite direction will interfere with the promoter expressing the forward direction.

Figure 7: Diagram of Register 0 Plasmid

According to previous experiments, the distance between the promoter and the start codon should be as close as possible, so we placed the necessary elements of the inducible promoter such as LacI, rhaS, rhaR outside the recombinase sites and only placed these promoter in the register region.

Figure 8: Map of Register 0 plasmid

After the plasmid was successfully constructed, we transferred the Register 0 plasmid into BL21 (DE3) competent cells and induced them by adding 1mM IPTG and 0.3% rhamnose at a final concentration after the cells grew to the logarithmic growth phase. Then we recorded the time span and fluorescence intensity as output. The results are as follows.

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

The single Register 0 without recombinase input can be an excellent AND gate. The figure above shows that IPTG and RHA have a relatively ideal induction effect, indicating that our promoters work properly. 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 promoting the transcription of mRNA when the rhamnose promoter is working.

qPCR

We used qPCR to verify whether the expression of these recombinases can modify the register plasmid made up of overlapping and orthogonal recombinase recognition sites. Here, we used A118 recombinase as an example. Firstly, we inserted the A118 recombinase gene in plasmid pET28a(+) and transformed the A118-pET28a(+) plasmid and Register 0 plasmid into E.coli BL21 (DE3) competent cells together. Then we picked a single colony, expanded the culture, added IPTG to a final concentration of 1mM, and induced at 37° for four hours. After plasmid extraction, we used the extracted plasmid samples for qPCR.

Figure 10. qPCR results of Register 0

In the Register 0 plasmid, our recombinase recognition sequence is in the reverse direction, and the recombinase will invert the fragment between the recombinase binding sites. We designed two qPCR primers in the same direction, located between and outside the recognition sites, so only the inverted plasmid can produce a signal. Our results confirm the feasibility of recombinase regulation of Register sequences.

Directed evolution

According to previous experiments, the recombination efficiency of some recombinases still needs to be improved. Therefore, we took A118 as an example and designed a continuous directed evolution system based on eMutaT7 to enhance its efficiency. More details about our design are inthe design part.

In order to verify the success of our directed evolution system, we firstly transformed the eMutaT7 plasmid, target plasmid, and selection plasmid into BL21 competent cells together. 100 μg/ml ampicillin, 50 μg/mL Kanamycin, 35 μg/ml chloramphenicol, 0.2% arabinose, and 0.1 mM IPTG were added into LB Agar plate, colonies were observed under intense UV light.

Figure 11. colonies observed under intense UV light

In the image above, we can clearly see fluorescence in the colony, indicating that eMutaT7 is effectively functioning and facilitating the expression of recombinases from the Target plasmid. A118 recombinase inverts the gfp fragment on the selection plasmid to activate the gfp gene. If time permits, we plan to proceed with our directed evolution experiments.