Difference between revisions of "Part:BBa K4891007"
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===Usage and Biology===
 
===Usage and Biology===
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<h3>1 PCR verification</h3>
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Colony PCR results show that aroK and aroL genes have been knocked out from the genome of the host strain (Figures 1-2), and the recombinant strains are named as strains E. coli YCY2-E. coli YCY3, respectively.
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{|
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4891/wiki/parts/6.png" ></html>
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|-
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| '''Figure 1 Knock out of aroK gene.'''
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|}
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The DNA fragment of knocked out and un-knocked out for aroK gene are 1800 bp and 2322 bp, respectively.
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{|
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| <html><img style="width:460px" src="https://static.igem.wiki/teams/4891/wiki/parts/7.png" ></html>
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|-
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| '''Figure 2 Knock out of aroL gene.'''
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|}
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The DNA fragment of knocked out and un-knocked out for aroL gene are 1800 bp and 2325 bp, respectively.
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<h3>2 Growth assay</h3>
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To evaluate whether the elimination of shikimate catabolism resulted in an auxotroph for amino acids, the growth curve in strains E. coli YCY1-YCY3 is examined. As seen in Figure 3, the growth rate and glucose consumption of strain E. coli YCY2-3 are significantly slowed down without the addition of L-tyrosine, L-phenylalanine, and L-tryptophan. In contrast, the growth status of these strains is improved after supplementing aromatic amino acids.
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{|
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4891/wiki/parts/8.png" ></html>
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|-
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| '''Figure 3 Growth curve in the engineered strains without (left) or with amino acid (right).'''
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|}
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WT (Escherichia coli MG1655), YCY1 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta), YCY2 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta ΔaroK), YCY3 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta ΔaroK ΔaroL)
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<h3>3 Shikimic acid biosynthesis</h3>
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To determine the effect of this composite component on the synthesis yield of SA, we used YCY4 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta; pTrcHisA-aroG-aroB-aroD-aroE) and YCY6 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta ΔaroK ΔaroL; pTrcHisA-aroG-aroB-aroD-aroE) for comparison. Due to the knockout of aroG and aroL, YCY6 increased SA yield by nearly 35 times compared to YCY4, reaching 0.75g/L. Overall, the composite component has the function of increasing the yield of SA.
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{|
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4891/wiki/parts/9.png" ></html>
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|-
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| '''Figure 4 Shikimate biosynthesis in the engineered strains. (96 h)'''
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|}
  
 
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Revision as of 08:27, 8 October 2023


aroL

With the disruption of shikimate catabolism, shikimate production could be improved.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 425
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 425
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 215
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 425
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 425
  • 1000
    COMPATIBLE WITH RFC[1000]