Composite

Part:BBa_S04055

Designed by: Robert Ovadia   Group: Guests   (2008-08-10)

Synthetic lacYZ operon

Construction intermediate


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.

2015CityU HK BBa S04055 qRTPCR.png

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.


2015CityU HK S04055 WesternBlot.png


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.

2015CityU HK BBa S04055 Characterization ONPGAssay.png

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

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 823
  • 1000
    COMPATIBLE WITH RFC[1000]


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