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 3. 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.
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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 3). 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 4) and is in agreement with the lacZ mRNA results described in Figure 3.
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Figure 4. 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.
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Figure 5. 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.
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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 5 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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Characterization of Lysis Gene Cassette Smutλ-Rλ-Rzλ (BBa_K1695038)
Upon induction with 0.2 mM, 1 mM and 5 mM of IPTG, recombinant E. coli harboring the lysis cassette Smutλ-Rλ-Rzλ (BBa_K1695038) showed a sharp drop in absorbance (A600) after 15 minutes whereas cells without IPTG induction showed continued as indicated by the steady increase in OD (Figure 6A). The results show that 0.2 mM IPTG is sufficient to initiate cell lysis after 15 minutes, and the speed of cell lysis is not increased at higher concentrations of IPTG (Figure 6B). Overall, the use of (1) OD600 measurement to determine cell growth or cell lysis (panel A) and (2) plate count (cfu; panel B) showed that the Lysis Gene Cassette is induced by IPTG and resulted in the initiation of cell lysis after 15 minutes of IPTG induction. Cells were completely lysed by 20 minutes. It would be interesting in future studies to determine if the time of cell lysis could be regulated by IPTG at concentrations below 0.2 mM.
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Figure 6. Cell lysis upon induction of lysis cassette by IPTG in E. coli cells. Recombinant E. coli carrying lysis cassette Smutλ-Rλ-Rzλ (BBa_K1695038) was cultured in minimal medium (supplemented with 0.2% glucose and 0.5(0.02%) casamino acid). IPTG at various concentrations (0 mM, ·) (0.2 mM, ·), 1 mM, ·) (5 mM, ·) was added to the bacterial culture at OD600 ~ 0.6. Samples were taken at 5-min and 10-min intervals from IPTG induced and uninduced cultures for (A) OD600 measurements and (B) cell plating on LB solid medium (CFU count) to determine the percentage (%) of cell survival, respectively.