In this project, I engineered, expressed, and analyzed recombinant fluorescent proteins — enhanced green fluorescent protein (eGFP) and an engineered blue variant (eBFP) — using contemporary molecular biology, microbiology, and protein biochemistry techniques. This end-to-end workflow provided hands-on experience with cloning, mutagenesis, bacterial expression, purification, and fluorescent visualization.

 

Subcloning the eGFP gene

• I amplified the eGFP open reading frame (ORF) from the pEGFP plasmid using PCR primers containing engineered restriction-enzyme overhangs. This allowed the insert to be directionally cloned into the chosen expression vector.

 

Constructing the expression plasmid

• The PCR product was ligated into the E. coli expression vector pQE30, which carries an N-terminal 6×His tag for affinity purification. The resulting pQE30/eGFP construct was transformed into competent XL1-Blue E. coli and plated on selective agar.​

 

Verifying the cloned construct and transformation

• Plasmid DNA was extracted from transformants, digested with restriction enzymes, and analyzed by agarose gel electrophoresis to confirm successful cloning and correct insert size.

​

Expressing recombinant His-tagged eGFP

• Induction with IPTG triggered expression of the His-tagged eGFP in E. coli. Cells were harvested and lysed for downstream purification.

​

Purifying and analyzing the protein

• His-tagged eGFP was purified using immobilized metal affinity chromatography (IMAC), then assessed by SDS-PAGE and Western blotting to verify purity and identity.

​

Engineering an enhanced blue fluorescent protein (eBFP)

• Using the cloned pQE30/eGFP construct as a template, I introduced a key mutation (Tyr66→His) through Phusion™ site-directed mutagenesis to generate eBFP. The linear PCR product was gel-purified, phosphorylated, ligated, and transformed into competent E. coli cells. Colonies expressing eBFP were examined under a UV transilluminator to observe the shift in fluorescence.