Difference between revisions of "Part:BBa K4891006"
Line 17: | Line 17: | ||
{| | {| | ||
− | | <html><img style="width: | + | | <html><img style="width:460px" src="https://static.igem.wiki/teams/4891/wiki/parts/7.png" ></html> |
|- | |- | ||
| '''Figure 2 Knock out of aroL gene.''' | | '''Figure 2 Knock out of aroL gene.''' |
Revision as of 08:26, 8 October 2023
aroK
With the disruption of shikimate catabolism, shikimate production could be improved.
Usage and Biology
1 PCR verification
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.
![]() |
Figure 1 Knock out of aroK gene. |
The DNA fragment of knocked out and un-knocked out for aroK gene are 1800 bp and 2322 bp, respectively.
![]() |
Figure 2 Knock out of aroL gene. |
The DNA fragment of knocked out and un-knocked out for aroL gene are 1800 bp and 2325 bp, respectively.
2 Growth assay
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.
![]() |
Figure 3 Growth curve in the engineered strains without (left) or with amino acid (right). |
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)
3 Shikimic acid biosynthesis
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.
![]() |
Figure 4 Shikimate biosynthesis in the engineered strains. (96 h) |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 200