The goal of this experiment was to evaluate the success of Golden Gate Assembly (GGA) constructs and assess gene expression through colony phenotype (red, green, or colorless) and PCR verification. The results indicate partial success of assembly, with clear evidence of transformation but inconsistent expression of the RFP gene.
From the transformation plates, colonies were successfully obtained across multiple conditions, confirming that the ligation and transformation steps were functional. The presence of green fluorescent colonies (GFP) and red colonies (RFP) in control groups supports that the reporter systems were capable of expression under proper conditions. However, in the experimental group (P5-33A), colonies were primarily colorless or green, with little to no red fluorescence observed. This suggests that RFP expression was significantly reduced or absent in this construct.
Quantitatively, the ratio of red to green colonies (~0.43 for P5-33A) was lower compared to control groups (~0.54–0.57), reinforcing that the experimental construct exhibited reduced RFP output relative to GFP. This imbalance indicates either:
inefficient assembly of the RFP-containing fragment,
improper insertion orientation, or
regulatory issues affecting transcription or translation of the RFP gene.
PCR analysis (as sketched in the gel diagram) further supports these observations. The expected band at ~196 bp (successful assembly) was present, while the 276 bp band (unsuccessful or incorrect assembly) was absent in successful samples. This confirms that some correctly assembled constructs were produced, but PCR alone does not guarantee proper gene expression. Therefore, even though assembly occurred at the DNA level, functional expression of RFP was still compromised.
Several experimental factors may explain the poor RFP expression:
Promoter or regulatory inefficiency – The promoter driving RFP may have been weaker or improperly assembled.
Mutation or frame shift – Small sequence errors during assembly could disrupt RFP translation.
Competition between reporters – GFP expression may dominate due to stronger folding efficiency or expression kinetics.
Transformation variability – Differences in plasmid uptake or copy number could affect expression levels.
Protein maturation differences – RFP matures more slowly than GFP, which may result in underestimation of red colonies.
Additionally, the presence of colorless colonies suggests either:
non-recombinant plasmids,
incomplete constructs, or
failure in reporter gene expression altogether.
Overall, while the experiment demonstrates successful DNA assembly and transformation, it highlights a key limitation: genotypic success does not always correlate with phenotypic expression. The P5-33A construct, in particular, showed poor RFP expression, indicating that further optimization—such as verifying sequence integrity, improving promoter design, or adjusting assembly conditions—is necessary.
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