4BBY1070 Genetics and Molecular Biology 2024-2025

Practical 2. Transformation of pGLO into Escherichia coli and analysis of GFP expression in
Escherichia coli containing pGLO using SDS-PAGE

Part A. Transformation of pGLO into Escherichia coli (10 marks)

Table 1. Transformation of pGLO into competent E. coli cells.

Observation of Observation of
Number of colonies
Agar medium colony morphology colony morphology
counted in daylight
in daylight under UV
Lawn of bacteria
Lawn of bacteria
LB 1 (Lawn of bacteria) No green
White in colour
fluorescence

No green
LB-amp 112 White in colour
fluorescence

LB-amp-ara 172 White in colour Fluorescent green

Part A. Results and discussion.

Three agar media were used: Luria-Bertani (LB) as a control, LB containing ampicillin
(LB-amp), and LB containing ampicillin and arabinose (LB-amp-ara). Table 1 compares
observations of the colonies in daylight and ultraviolet light (UV).

Bacteria in the LB medium appeared white in daylight but did not fluoresce under UV. This
medium contained substantially more colonies than the others due to a lack of ampicillin-induced
selective pressure.
LB-amp cultured colonies appeared white and did not exhibit green fluorescence under UV.
Fewer bacteria were observed on this plate, as the medium contained the antibacterial ampicillin,
which allowed only bacteria modified with pGLO plasmids (carrying the ampicillin-resistance
gene) to survive and form colonies.

Colonies on the LB-amp-ara plate also appeared white, with a similar number of colonies to the
LB-amp plate as ampicillin remained present. However, bacteria in this medium exhibited green
fluorescence under UV. The presence of arabinose triggered the araC gene to change the
conformation of araC binding protein, enabling RNA polymerase to bind to the araBAD
promoter region. RNA polymerase consequently transcribed the GFP gene (located downstream
of araBAD region), resulting in GFP expression, thus the colonies fluoresce under UV.

The other mediums lacked arabinose, preventing the araC protein from changing shape, therefore
inhibiting GFP transcription and fluorescence.

Overall, arabinose triggers GFP expression, causing fluorescence under UV, while ampicillin
selects for resistant bacteria, reducing colony count compared to non-selective LB.

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Part B. Analysis of GFP expression in Escherichia coli containing pGLO using SDS-PAGE
(40 marks)

Figure 1: SDS-PAGE gel showing five lanes of protein separation according to molecular
weights labeled in kilodaltons (kD). Lane 4 displays a distinct green fluorescent protein (GFP)
band around 34 kD, indicating GFP presence.
Table 2. Distance Bio-Rad Precision Plus Protein Unstained Standard markers have moved from
the line (measured from Figure 1).

Bio-Rad Precision Plus

Distance moved from
Protein Unstained
well (mm)
Standard
250 kD 12
150 kD 15
100 kD 22
75 kD 29
50 kD 40
37 kD 47
25 kD 64
20 kD 70
15 kD 84
 10 kD 95

Table 3. The experimental size of green fluorescent protein from SDS-PAGE analysis.

Lane number on Distance moved Experimental size

Figure 1 from well (mm) (kD)
Lane 52 34

Figure 2. Graph depicting relationship between the Bio-Rad Precision Plus Protein Unstained
Standard size (kD) and distance (mm) migrated from well
Part B. Results and discussion.

Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) was conducted to

analyze the expression and stability of GFP in E. coli modified with the pGLO plasmid. Figure 1
shows protein separation across five lanes. Table 2 provides migration distances of standard
proteins, and Table 3 estimates the experimental size of GFP.

Lane 1 contains the molecular weight marker, with bands corresponding to known protein
weights. This is used as a reference to estimate protein sizes in other lanes, particularly GFP.

Lane 2 contains bacterial lysate grown in the LB-amp medium without heat treatment. Multiple
bands representing cellular proteins are visible, but no fluorescent band is detected, indicating
that GFP was not expressed under these conditions due to the absence of arabinose.

Lane 3 contains bacterial lysate cultured in LB-amp medium and subjected to heat treatment at
95°C. Protein denaturation is evident, as the bands appear diffused due to increased
fragmentation. No fluorescence is present, suggesting that GFP was either not expressed or was
degraded by heat. It was most likely not expressed, as GFP, the culture medium, did not contain
arabinose.

Lane 4 contains E. coli lysate cultured in the LB-amp-ara medium without heat treatment. A
fluorescent band at 34 kD is visible, demonstrating GFP expression. The prominence of the band
suggests successful GFP production under these conditions.

Lane 5 contains lysate from E. coli cultured in the LB-amp-ara medium and heat-treated at 95°C.
While a faint protein band is visible at the expected GFP size, fluorescence is absent, indicating
that heat treatment denaturation disrupted the GFP structure. This supports the known
heat-sensitivity of GFP, as fluorescence depends on its folded structure, which is disrupted at
high temperatures.
The results confirm that GFP was present but is expressed only in the presence of arabinose.
Heat treatment is shown to denature the GFP protein, preventing fluorescence under UV. The
lack of fluorescence in heat-treated samples demonstrates GFP's reliance on folding for function.
These findings validate the regulation of GFP expression by the araC operon and demonstrate
temperature sensitivity.
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Appendix A (no marks)

Figure 3.

Figure 3 shows the same results as Figure 1 so all steps were performed correctly