Difference between revisions of "Part:BBa K4879008"

 
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<partinfo>BBa_K4879008 short</partinfo>
 
<partinfo>BBa_K4879008 short</partinfo>
  
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The transcriptional unit coding for CvFAP, designed for expression in <i>Y. lipolytica</i>.
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The expression cassette was assembled together along with the JcFATB and the JcFATA expression cassettes with the help of the NEBuilder HiFi DNA Assembly kit.
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===Characterization===
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<b>Cloning:</b>
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The assembled gene(s) were then inserted into the pYLEX plasmid (sourced from the Yarrowia Lipolytica Expression Kit, procured from Yeastern Biotech), by the means of NEBuilder HiFi DNA Assembly. The expected clones were then digested with NotI and SalI. The resulting digestion product is run on a 0.8% agarose gel. The cloning was confirmed, with the cloning success also being verified with sequencing. The linearized fragment was expected to be at above the 10 kbp band in the ladder (~13,000 bp)
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<html><img src="https://static.igem.wiki/teams/4879/wiki/bba-k4879000-gel.jpg"width="720"height="720"></html>
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Fig: Lanes 1-4 contain the possible clones and lane 5 contains the ladder. Clones in lanes 1,3, and 4 are likely successful clones.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 15:59, 11 October 2023


CvFAP Expression Construct

The transcriptional unit coding for CvFAP, designed for expression in Y. lipolytica.

The expression cassette was assembled together along with the JcFATB and the JcFATA expression cassettes with the help of the NEBuilder HiFi DNA Assembly kit.

Characterization

Cloning: The assembled gene(s) were then inserted into the pYLEX plasmid (sourced from the Yarrowia Lipolytica Expression Kit, procured from Yeastern Biotech), by the means of NEBuilder HiFi DNA Assembly. The expected clones were then digested with NotI and SalI. The resulting digestion product is run on a 0.8% agarose gel. The cloning was confirmed, with the cloning success also being verified with sequencing. The linearized fragment was expected to be at above the 10 kbp band in the ladder (~13,000 bp)

Fig: Lanes 1-4 contain the possible clones and lane 5 contains the ladder. Clones in lanes 1,3, and 4 are likely successful clones.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 2320
    Illegal suffix found in sequence at 2590
    Illegal EcoRI site found at 2584
    Illegal PstI site found at 790
    Illegal PstI site found at 1222
    Illegal PstI site found at 1489
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2320
    Illegal EcoRI site found at 2584
    Illegal SpeI site found at 2591
    Illegal PstI site found at 790
    Illegal PstI site found at 1222
    Illegal PstI site found at 1489
    Illegal PstI site found at 2605
    Illegal NotI site found at 2326
    Illegal NotI site found at 2598
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2320
    Illegal EcoRI site found at 2584
    Illegal XhoI site found at 1955
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 2320
    Illegal suffix found in sequence at 2591
    Illegal EcoRI site found at 2584
    Illegal PstI site found at 790
    Illegal PstI site found at 1222
    Illegal PstI site found at 1489
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 2320
    Illegal EcoRI site found at 2584
    Illegal XbaI site found at 2335
    Illegal SpeI site found at 2591
    Illegal PstI site found at 790
    Illegal PstI site found at 1222
    Illegal PstI site found at 1489
    Illegal PstI site found at 2605
    Illegal NgoMIV site found at 2110
    Illegal AgeI site found at 2086
  • 1000
    COMPATIBLE WITH RFC[1000]