Synthetic lacYZ operon
Contribution (CityU_HK 2015)
Group: CityU_HK, 2015
Authors: Chan Ka Lung, Yeung Pak Pui
Summary: Improved/expanded characterization of LacY-LacZ Operon
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.
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.
B. Western Blot analysis
Western 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.
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.
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.
Figure 3. 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.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 823
- 1000COMPATIBLE WITH RFC