Difference between revisions of "Part:BBa K5298000"

 
 
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<h1><b>Tongji-China 2024: Induction of xylE expression color gradient by the maltose operon</b></h1>
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The genes of interest, xylE and GUSA, were synthesized by Genewiz and returned as plasmids with the pUC-GW-Kan vector. We plan to use the ClonExpress technology for homology-directed recombination to construct the plasmids. To achieve this, we have designed four sets of primers, with two sets specifically incorporating homology arms.
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After performing the PCR reactions, we conducted agarose gel electrophoresis to verify the PCR products. The two bands observed on the gel were of the expected sizes. We then proceeded to excise the bands, purify the DNA fragments, and perform the ligation reaction using the ClonExpress technology for seamless cloning.
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After sequencing and validating the recombinant plasmids, we confirmed the success of the experiments. Building upon the experience gained from the expression experiments with the bFMO-pET28a plasmid, we also performed OD600 measurements and plotted logarithmic growth curves for the yellow and red plasmids to investigate the optimal time during which the engineered bacteria are in the logarithmic growth phase. Following the addition of the inducer, the bacteria were cultured in liquid LB medium at 37°C for 16 hours, and we observed the expression of visible colors.
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Figure 8: Color Expression in Liquid Medium
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Furthermore, we also conducted OD600 growth curve measurements for these two engineered bacterial strains and found that their growth profiles were generally consistent with those of the blue plasmid in the DE3 strain.
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1. Characterization in Liquid Medium
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Visualization of Changes in Yellow Color Over Time in Liquid Medium
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Changes in Solution Purity and Brightness Over Time in Liquid Medium
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Note: The lower the brightness, the darker the color; the higher the purity, the more yellow the color.
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2. Characterization in Solid Medium
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Variation of Yellow Intensity Under Different Conditions
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Box Plot of Univariate Analysis

Latest revision as of 06:06, 2 October 2024


xylE gene

This gene encodes the enzyme catechol-2,3-dioxygenase (metapyrocatechase), which converts catechol to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde. This is a useful reporter gene; colonies or broths expressing active XylE, in the presence of oxygen, will rapidly convert catechol, a cheap colourless substrate, to a bright yellow compound with an absorbance maximum around 377 nm.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 311
    Illegal NgoMIV site found at 483
    Illegal AgeI site found at 834
  • 1000
    COMPATIBLE WITH RFC[1000]


Tongji-China 2024: Induction of xylE expression color gradient by the maltose operon

The genes of interest, xylE and GUSA, were synthesized by Genewiz and returned as plasmids with the pUC-GW-Kan vector. We plan to use the ClonExpress technology for homology-directed recombination to construct the plasmids. To achieve this, we have designed four sets of primers, with two sets specifically incorporating homology arms.
After performing the PCR reactions, we conducted agarose gel electrophoresis to verify the PCR products. The two bands observed on the gel were of the expected sizes. We then proceeded to excise the bands, purify the DNA fragments, and perform the ligation reaction using the ClonExpress technology for seamless cloning.

After sequencing and validating the recombinant plasmids, we confirmed the success of the experiments. Building upon the experience gained from the expression experiments with the bFMO-pET28a plasmid, we also performed OD600 measurements and plotted logarithmic growth curves for the yellow and red plasmids to investigate the optimal time during which the engineered bacteria are in the logarithmic growth phase. Following the addition of the inducer, the bacteria were cultured in liquid LB medium at 37°C for 16 hours, and we observed the expression of visible colors.

Figure 8: Color Expression in Liquid Medium
Furthermore, we also conducted OD600 growth curve measurements for these two engineered bacterial strains and found that their growth profiles were generally consistent with those of the blue plasmid in the DE3 strain.
1. Characterization in Liquid Medium

Visualization of Changes in Yellow Color Over Time in Liquid Medium

Changes in Solution Purity and Brightness Over Time in Liquid Medium
Note: The lower the brightness, the darker the color; the higher the purity, the more yellow the color.
2. Characterization in Solid Medium

Variation of Yellow Intensity Under Different Conditions 

Box Plot of Univariate Analysis