Difference between revisions of "Part:BBa K3402055"
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===Usage and Biology===
 
===Usage and Biology===
  
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up<i>PXA1</i> and do<i>PXA1</i> are homologous arms, which means this device will edit the <i>PXA1</i> site. The β-Rec/six self-excising system will help to recycle the hygromycin resistance gene. <i>UGTB</i> is the key gene in the synthesis of sophorolipid.
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up<i>PXA1</i> and do<i>PXA1</i> are homologous arms, which means this device will edit the <i>PXA1</i> site. The β-Rec/six self-excising system will help to excise the hygromycin resistance gene. UDP-glucosyltransferase B (<i>UGTB</i>) is the key gene in the synthesis of sophorolipids.
 
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When we add this fragment to the Cas9 expression device, we will use promoters with different expression strength as we characterized before to control the expression level of <i>UGTB</i>. Then we can produce different amount of lactone-type sophorolipids.
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Choose <i>PXA1</i> as the gene editing site. Three strong promoters P<i>tef1</i>, P<i>eno</i> and P<i>gki</i> were knocked in <i>PXA1</i> site to over-express UDP-glucosyltransferase B (<i>UGTB</i>).
 
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When the over-expression of <i>SBLE</i> and <i>UGTB</i> both work in the cell, there will be two type of sophorolipids produced by our engineering yeast, acid type and lactone type. Different types of sophorolipids have different functions. The combination of lactone type and acid type may increase the effect of sophorolipid. So, we can control the ratio of lactone and acid type sophorolipids.
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Then we transformed the corresponding over-expression fragments and the Cas9 and sgRNA expression cassette to the wild-type <i>Starmerella bombicola</i>.
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After incubation and fermentation, test the yield and acid/lactone ratio of sophorolipids produced by different strains.
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After over-expressing <i>UGTB</i>, the yield could be increased a lot than that of the control.
  
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[[Image:Table-The yield of sophorolipids produced by different strains.jpeg|500px|thumb|center|'''Table. 1''' The yield of sophorolipids produced by different strains]]
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[[Image:Chart-The yield of sophorolipids by over-expression of UGTB.png|500px|thumb|center|'''Chart.1''' The yield of sophorolipids by over-expression of UGTB
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('''W: '''wild type; '''S1: '''::Ptef1-UGTB; '''S2: '''::Peno-UGTB; '''S3: '''::Pgki-UGTB)]]
  
 
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Revision as of 18:08, 27 October 2020


Over-expression UGTB cassette

This device is composed of 50bp-upPXA1(BBa_K3402037), Six site(BBa_K3402032), Pgalk(BBa_K3402033), Rec(BBa_K3402034), Tgki(BBa_K3402019), hph(BBa_K3402012), Ptef1(BBa_K3402007), UGTB(BBa_K3402011), Tsyn7(BBa_K3402001), 50bp-doPXA1(BBa_K3402038).

Over-expression UGTB cassette.png

Usage and Biology

upPXA1 and doPXA1 are homologous arms, which means this device will edit the PXA1 site. The β-Rec/six self-excising system will help to excise the hygromycin resistance gene. UDP-glucosyltransferase B (UGTB) is the key gene in the synthesis of sophorolipids.
Choose PXA1 as the gene editing site. Three strong promoters Ptef1, Peno and Pgki were knocked in PXA1 site to over-express UDP-glucosyltransferase B (UGTB).
Then we transformed the corresponding over-expression fragments and the Cas9 and sgRNA expression cassette to the wild-type Starmerella bombicola.
After incubation and fermentation, test the yield and acid/lactone ratio of sophorolipids produced by different strains. After over-expressing UGTB, the yield could be increased a lot than that of the control.

Table. 1 The yield of sophorolipids produced by different strains
Chart.1 The yield of sophorolipids by over-expression of UGTB
(W: wild type; S1: ::Ptef1-UGTB; S2: ::Peno-UGTB; S3: ::Pgki-UGTB)

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 1076
    Illegal BglII site found at 5893
    Illegal BamHI site found at 3479
    Illegal XhoI site found at 1910
    Illegal XhoI site found at 4631
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 219
    Illegal AgeI site found at 5315
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5474
    Illegal BsaI.rc site found at 5429