Composite

Part:BBa_K3909018

Designed by: Xixi Song   Group: iGEM21_Worldshaper-Nanjing   (2021-10-14)
Revision as of 10:36, 16 October 2021 by SylviaSong (Talk | contribs)


pYLXP-ylPOT1-ylMFE1-ylPOX1

This composite part consist of pYLXP’-ylPOT1 (BBa_K3909010), pYLXP’-ylMFE1 (BBa_K3909011) and pYLXP’-ylPOX1 (BBa_K3909012). As shown in figure 1, our biobricks design is mainly divided into two parts: fatty acid degradation and γ-linolenic acid synthesis. The BBa_K3909018 is belong to part one --fatty acid degradation.

Fig.1 The overview of our biobricks design.

We plan to enhance the oli degradation pathway by expressing three endogenous fatty acid degradation genes ylMEF1 (BBa_K3909006), ylPOT1 (BBa_K3909007), and ylPOXn (from BBa_K3909000 to BBa_K3909005), which are related to the metabolim of transforming acyl-CoA into acetyl-CoA in peroxisome (β-oxidation). Specifically, the β-oxidation includes three steps: i) oxidation, that catalyzed by six acyl-CoA oxidases (translated from ylPOX1 to ylPOX6); ii) hydration and dehydration, that catalyzed by multifunctional enzyme (translated from ylMFE1); and iii) thiolysis, that catalyzed by 3-ketoacyl-CoA thiolase (translated from ylPOT1)[1]. Firstly,we constructed the single gene-overexpressed plasmids by the aforementioned method, including pYLXP’-ylPOT1(BBa_K3909010), pYLXP’-ylMEF1 (BBa_K3909011) and pYLXP’-ylPOXn (from BBa_K3909012 to BBa_K3909017) , which are shown in Figure 2.

Fig.2 Single fragment plasmid construction.

For overpressing the whole oil degradation pathway, we further combined these genes by BioBrick method, and constructed plasmids pYXLP’-ylPOT1-ylMFE1-ylPOXn (from BBa_K3909018 to BBa_K3909023), as shown in figure 3. The Y. lipolytica (Po1f) we used cannot synthesize leucine by itself, so the Leu2 marker on the plasmid can be used as marker gene. Then, the constructed plasmids were transformed into Y. lipolytica Po1f.

Fig.3 Multi-fragment plasmid construction.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1343
    Illegal BamHI site found at 6611
    Illegal BamHI site found at 6822
    Illegal XhoI site found at 164
    Illegal XhoI site found at 565
    Illegal XhoI site found at 2378
    Illegal XhoI site found at 2840
    Illegal XhoI site found at 4249
    Illegal XhoI site found at 4341
    Illegal XhoI site found at 5161
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 832
    Illegal NgoMIV site found at 925
    Illegal NgoMIV site found at 1159
    Illegal NgoMIV site found at 7653
    Illegal NgoMIV site found at 7705
    Illegal AgeI site found at 1657
    Illegal AgeI site found at 1741
    Illegal AgeI site found at 1893
    Illegal AgeI site found at 2791
    Illegal AgeI site found at 4410
    Illegal AgeI site found at 5115
    Illegal AgeI site found at 5642
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2806
    Illegal BsaI site found at 4746
    Illegal BsaI site found at 5301
    Illegal BsaI site found at 7337
    Illegal BsaI.rc site found at 2
    Illegal BsaI.rc site found at 844
    Illegal BsaI.rc site found at 2216
    Illegal BsaI.rc site found at 4723
    Illegal BsaI.rc site found at 5400
    Illegal BsaI.rc site found at 5965
    Illegal SapI site found at 4346
    Illegal SapI site found at 5501


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