Difference between revisions of "Part:BBa K3909010"
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Firstly, we amplified the sequences of genes ylPOT1, ylMFE1, ylPOX1, ylPOX2, ylPOX3, ylPOX4, ylPOX5 , and ylPOX6 with using the Y. lipolytica as the template by PCR method. The result of genes amplification has been showed in Figure 2.  
 
Firstly, we amplified the sequences of genes ylPOT1, ylMFE1, ylPOX1, ylPOX2, ylPOX3, ylPOX4, ylPOX5 , and ylPOX6 with using the Y. lipolytica as the template by PCR method. The result of genes amplification has been showed in Figure 2.  
  
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[[File:Engineering.Fig1wsnj.png|600px|thumb|center| Fig.2 Amplifying the fragments of genes ylPOX1 (a), ylPOX2 (b), ylPOX3 (c), ylPOX4 (d), ylPOX5 (e), ylPOX6 (f), ylPOT1 (g), and ylMFE1 (h).]]
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[[File:Fig.2-wsnj.png|600px|thumb|center| Fig.2 Amplifying the fragments of genes ylPOX1 (a), ylPOX2 (b), ylPOX3 (c), ylPOX4 (d), ylPOX5 (e), ylPOX6 (f), ylPOT1 (g), and ylMFE1 (h).]]
  
 
===2. Linearizing plasmid pYLXP’===
 
===2. Linearizing plasmid pYLXP’===

Revision as of 10:10, 16 October 2021


pYLXP-ylPOT1

A composite part consist of pTEF promoter (BBa_K3909008 ), ylPOT1 (BBa_K3909007) and XPR2_terminator (BBa_K3909009).

Our goal is to improve the cell growth of Y. lipolytica and convert a large amount of gutter oil to γ-linolenic acid when Y. lipolytica grows with gutter oil as the sole carbon source . 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 1.

Fig.1 Single 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 XhoI site found at 164
    Illegal XhoI site found at 565
  • 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 AgeI site found at 1657
    Illegal AgeI site found at 1741
    Illegal AgeI site found at 1893
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2
    Illegal BsaI.rc site found at 844


Usage and Biology

Results: Constructing recombinant plasmids for overexpressing the β-oxidation pathway

1. PCR amplification of genes

Firstly, we amplified the sequences of genes ylPOT1, ylMFE1, ylPOX1, ylPOX2, ylPOX3, ylPOX4, ylPOX5 , and ylPOX6 with using the Y. lipolytica as the template by PCR method. The result of genes amplification has been showed in Figure 2.

File:Fig.2-wsnj.png
Fig.2 Amplifying the fragments of genes ylPOX1 (a), ylPOX2 (b), ylPOX3 (c), ylPOX4 (d), ylPOX5 (e), ylPOX6 (f), ylPOT1 (g), and ylMFE1 (h).

2. Linearizing plasmid pYLXP’

The YaliBrick plasmid pYLXP’ was used as the expression vector in this project. Plasmid constructions were performed by using preciously reported methods[2]. For linearizing plasmid, we used the nuclease SnaBI and KpnI to digest plasmid pYLXP’. The result of plasmid pYLXP’ digestion has been showed in Figure 3.