Team:CityU HK/Results
Characterization of the lactose-inducible promoter (BBa_K1695053)
The GFP reporter biobrick (BBa_K1695053) was constructed by linking the above lacI-regulated promoter (BBa_K1695000) to GFP (BBa_I13504) (Figure 1). The response of this GFP reporter construct to IPTG induction was then measured based on the GFP fluorescence signal (excitation wavelength: 485nm; emission wavelength: 520 nm). GFP fluorescence signal from cells carrying the PL8-UV5-GFP construct (BBa_K1695053) was monitored over a 4.5-hour time course under a range of IPTG concentrations ranging from 0.01 – 10 mM. Normalization of GFP fluorescence was performed based on the A600 values of the culture.
Figure 1. Construct of the reporter biobrick, BBa_K1695053
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Figure 2. Expression analysis of L8-UV5 promoter-GFP biobrick (BBa_K1695053). (A) Activation of GFP expression in the L8-UV5 promoter construct is dependent on IPTG concentrations below 1 mM. (B) Fluorescence response of the L8-UV5 promoter-GFP gene construct (BBa_K1695053) was measured over a 4.5-h time course under a range of IPTG concentrations (from 0.01 mM to 10 mM). Results show that induction of the L8-UV5 promoter is independent of IPTG at concentrations above 1 mM, where LacI is saturated and promoter is fully turned on.
Results in Figure 2 show that GFP expression in the L8-UV5 promoter-GFP construct is dependent on IPTG concentrations below 1 mM. At IPTG concentrations above 1 mM, expression of GFP has reached a maxima regardless of the sampling time points.
Results in Figure 2 show that GFP expression in the L8-UV5 promoter-GFP construct is dependent on IPTG concentrations below 1 mM. At IPTG concentrations above 1 mM, expression of GFP has reached a maxima regardless of the sampling time points.
Improved/ expanded characterization of LacY-LacZ biobrick (BBa_S04055)
Figure 1. Quantitative RT-PCR analysis of lacY and lacZ expression in E. coli cells.
Expression of the lacZ and lacY genes was measured in control DH5α cells (BLUE) and BBa_S04055 recombinant DH5α cells (RED). Cells were cultured overnight in LB medium + antibiotic and total RNA harvested for qRT-PCR using 16S rRNA for normalization. |
A. Quantitative real-time PCR
Quantitative real-time PCR analysis showed that both lacY and lacZ mRNA transcripts are expressed at high levels in recombinant E. coli cells (harboring the BBa_S04055 biobrick) as compared to control DH5α cells (Figure 1). Expression of lacY and lacZ is 9- fold and 2-fold higher, respectively, in recombinant cells with respect to the control cells.
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B. Western Blot AnalysisWestern blot analysis showed that the β-galactosidase protein (LacZ) (indicated by the arrow) is expressed at a significantly higher level in recombinant E. coli cells (BBa_S04055) relative to the control (Figure 2) and is in agreement with the lacZ mRNA results described in Figure 1.
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Figure 2. Western Blot analysis of β-galactosidase (LacZ) protein.
Expression of the β-galactosidase protein (~ 135 kDa band) in control (Lane 1) and recombinant (BBa_S04055) (Lane2) E. coli cells. |
Figure 4. Expression of β-galactosidase as measured by the ONPG assay.
Control (BLUE) E. coli cells and BBa_S04055 recombinant cells (RED) at OD600=0.3 were harvested and lysed to release intracellular β-galactosidase, which was then measured for its activity by the ONPG assay. The conversion was measured by A420 at 15 minute interval. |
C. Measurement of β-galactosidase enzyme activity using ONPG Assay
The level of β–galactosidase activity was measured in recombinant (BBa_S04055) and control E. coli cells using the ONPG colorimetric assay. The results in Figure 3 show that the concentration of the cleavage product (A420) increased linearly within 60 minutes in the recombinant cells (BBa_S04055) while no change in absorbance was observed in control cells which indicated that the β-galactosidase enzyme activity is expressed in the recombinant cells and is in agreement with the results previously reported by the 2008 Caltech iGEM team.
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