Results
Achievements and future plans

FRET-EryK Results

🧬 Ligation

Our initial goal was to ligate the gene for ECFP-eryK-mVENUS from a pUC57 to a pET28b backbone. After several cycles of digestion, miniprep and ligation, the goal was finally achieved.

Figure 1 shows a colony PCR gel run with primers for the entire construct (ECFP-eryK-mVENUS) and primers for only the mVENUS protein. Controls were run for both of these to ensure the band we saw corresponded to the protein. The observed bands were at the expected weights for both samples and matched the controls loaded. This indicates that the pET28b plasmid contains the entire protein sequence and can be used for transformation.

Figure 1. Colony PCR results confirming the presence of the ECFP-EryK-mVENUS protein construct within the plasmid.

💫 Transformation

Once the colony PCR showed our insert correctly ligated to the plasmid backbone, we transformed into BL21 E. coli and plated. The control was BL21 E. coli transformed with an empty pET28b vector. Colonies were observed which was a good sign, however the real test was exposing both plates to UV.

Figure 2. Transformation of BL21 with A) ECPF-EryK-mVENUS and B) pET28b(+) empty vector. Both plates were exposed to UV light to confirm fluorescence.

Once both plates were placed on the UV transilluminator it was clear that the colonies transformed with the whole FRET system emitted a higher fluorescence signal than those with only the pET28b backbone (Figure 2). This indicates that the transformation was successful and our gene sequence is being translated.

✨ Overexpression

Having transformed E. coli with our FRET system, we could find the optimum conditions for overexpression. After testing several conditions, we induced at 16 ºC overnight, lyzed through sonication and measured intracellular protein presence and concentration through a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel showed a very faint band present at the appropriate weight for the ECFP-eryK-mVENUS.

Figure 3. SDS-PAGE gel (8 %) showing protein overexpression results for induced colonies.

AtPCS Results

🧬 Cloning of AtPCS into pET28b

Our initial goal was to clone the AtPCS gene from a pUC57 to a pET28b backbone. After several cycles of optimizing digestion, miniprep and ligations, the goal was finally achieved. Figure 4 and 5 show LB agar and Kanamycin, where 300 μL of the transformation reaction of E. coli TOP10 cells with the pET28b(+)_AtPCS plasmids were added, which confirmed that the cells acquired antibiotic resistance through the plasmid.

Figure 4. Transformation of pET28b(+)_AtPCS plasmid into E. coli TOP10 cells (1 hour ligation).
Figure 5. Transformation of pET20b(+)_AtPCS plasmid into E. coli TOP10 cells (O/N ligation).

Additionally, Figure 6 shows a colony PCR performed with five colonies from the previous plates to verify the presence of AtPCS gene in the pET28b(+) plasmids. Band was observed near the 1.5kbp mark, which matches the length of the gene (1461 bp).

Figure 6. Amplification of AtPCS gene from random colonies selected from the transformation of 1 hour ligation into E. coli TOP10 cells. The ladder used was the 1kb Plus ladder (Invitrogen) .

To identify, through an additional methodology, the clones that were correctly transformed with the gene, restriction assays were performed on plasmids obtained from colonies 1-3 using the NdeI and EcoRI-HF enzymes (New England Biolabs). Figure 7 shows the agarose gel (1 %) with the results of the enzymatic digestions, where a band corresponding to the length of the AtPCS gene was identified in sample from colony 1.

Figure 7. Digestion of pET28b(+) plasmid with NdeI and EcoRI-HF restriction enzymes. The ladder used was the 1kb Plus ladder (Invitrogen). A positive result is observed in the lane corresponding to plasmid from colony 1.

✨ Protein overexpression

Having transformed E. coli BL21 with pET28b(+)_AtPCS plasmid, we tested different conditions for overexpression. Finally, induction was carried out at 16 ºC overnight. Cell lysis was performed through sonication and intracellular protein presence and concentration was measured through a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as shown in Figure 8. The gel showed a very faint band present at the appropriate weight for the AtPCS gene (1461 bp).

Figure 8. SDS-PAGE gel (8 %) showing protein overexpression results for induced colonies. In wells corresponding to colonies 4-7 supernatants, a very faint blurred band near 55 kDa (AtPCS molecular weight) is thought to be seen.