The basis for the assembly of this composite part was the previous iteration of the biosensor, ECFP_EryK_mVenus(BBa K4447004), which was made up of the 3 genes in a pET-28b backbone. In order to successfully assemble the construct through Gibson Assembly without scars between parts and ensure proper expression of the full fusion construct, we amplified the vector using primers that bind to the ends of both fluorescent proteins and exclude the center EryK gene, obtaining an empty FRET backbone with the homology regions corresponding to the gene of interest, in this case TjPCs. This was the basis for the construction of the other two versions of the biosensor: ECFP_RifMo_mVenus (BBa_K5439003), and ECFP_IpfF_mVenus (BBa_K5439006). This method of assembly effectively makes our system modular and customizable, as the detector gene can be switched out to cover a wider range of contaminants.

Along with amplifying the FRET backbone, we amplified TjPCs (BBa_K5439001)with primers that generate homology regions corresponding to those generated by the backbone amplification. After several optimization cycles, which included optimizing the annealing temperature, number of cycles, and elongation times, we obtained purified fragments to use in the assembly. Figure 2 displays the PCR gels for both the vector and the gene.

With both fragments amplified and purified, we proceeded to assemble the construct through Gibson Assembly (NEB Gibson Assembly Master Mix). Table 1 shows the components used for the assembly reaction. Assembly was done with 100 ng of vector and a 3-fold molar excess of insert, and the reaction was incubated at 50 °C for 1 hour.

Table 1. Gibson Assembly reaction components

Reagent	Quantity
FRET backbone	2.3 µL
TjPCs	0.6 µL
Gibson Assembly Master Mix	5 µL
Nuclease-free water	2.1 µL

After the assembly, the next step was to transform the assembled ECFP_TjPCs_mVenus product into E. coli BL21, an expression strain. This step required optimization as well, particularly regarding the efficiency of our competent cells. After optimization, we successfully obtained transformed colonies containing our construct (Figure 3.)

Confirmation of construct insertion through restriction digestion

As a confirmation step, we performed minipreps on transformed colonies and digested the resulting plasmid with Nco I and XhoI , in order to ensure the transformed colonies contained the plasmid with the full construct. Table 2 shows the components used for the restriction digest, while Figure 4 shows the resulting gel, showing bands that correspond to the approximate full length of the ECFP_TjPCs_mVenus construct and the rest of pET28b.

Table 2. Restriction digest

Reagent	Quantity
Restriction Enzyme 10X Buffer	5 µL
DNA (1 μg/μL)	1 µL
NcoI restriction enzyme	1 µL
XhoI restriction enzyme	1 µL
BSA (10 μg/μL)	0.2 µL
Nuclease-free water	To 20 µL
Total Volume	20 µL

Protein expression

Once we had the certainty of a successful assembly and transformation in BL21, selected colonies were induced with 0.4 mM IPTG to stimulate protein overexpression and obtain the full ECFP_TjPCs_mVenus fusion construct. We attempted various temperatures and induction conditions, and the last trial at 16 °C for 16 hours yielded visible bands corresponding to the approximate molecular weight of the full construct (~106 kDa). As shown in Figure 5 , the bands corresponding to this weight were not visible in the negative controls, which were the same cells transformed with an empty pET28b backbone containing no insert. This shows evidence for the expression of our fusion construct. Further tests would include the purification of the protein and assays correlating the concentration of substrate to the fluorescence produced by the FRET system.

References