Part:BBa_K5439004

FRET-based system for the detection of cadmium

FRET-based sensor system for the detection of cadmium and other heavy metals that consists of phytochelatin synthase from Thlaspi japnonicum (BBa_K5439001),an enzyme that catalyzes the biosynthesis of phytochelatins using as a co-substrate the heavy metal cadmium, flanked by two fluorescent proteins: ECFP (BBa_K1159302)as an energy donor and mVenus (BBa_K1907000)as an energy acceptor.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 895
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 745
  Illegal BglII site found at 2154
  Illegal XhoI site found at 2176
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2825
  

Usage and Biology

Figure 1. Predicted structure with the best PAE obtained from ColabFold showing ECFP (green), EryK (blue), and mVenus (yellow).

Characterization

Gene Amplification, Assembly and Transformation

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.

Figure 2. (A) Agarose gel showing the amplified TjPCs gene used for the assembly of the construct. The band corresponds to the approximate length of the TjPCs gene (~1455 bp) (B) Agarose gel showing the amplification of the FRET backbone in pET28b. The faint band corresponds to the amplicon's expected length (~6800 bp), while the more visible band corresponds to unamplified plasmid in its supercoiled form.

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.)

Figure 3. ECFP_TjPCs_mVenus transformed colonies.

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

Figure 4. Gel corresponding to the digestion of plasmids from transformants with Nco I and XhoI . The ~3kbp band corresponds to the approximate length of the ECFP_TjPCs_mVenus construct, and the ~6kbp band corresponds to the rest of pET28b

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.

Figure 5. SDS-PAGE gel showing protein overexpression results of the full ECFP_TjPCs_mVenus fusion construct. The highlighted band corresponds to the approximate molecular weight of the construct. No band of the same molecular weight as the desired protein was observed in the negative control sample.

References