Difference between revisions of "Part:BBa K3578010"
(→‎1.Plasmid Construction)
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[[File:Figure 1 Schematic diagram of plasmid construction process.png|500px|thumb|center|Figure 1 Schematic diagram of plasmid construction process.]]  
 
[[File:Figure 1 Schematic diagram of plasmid construction process.png|500px|thumb|center|Figure 1 Schematic diagram of plasmid construction process.]]  
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By the PCR experiment, we randomly selected 10 strains in which AnGlu and Leu expression cassette have been successfully integrated into the genome. We designed the primers(F/R) to identify the Anglu expression cassette. The primer design was shown in the figure 2, and the predicted PCR result is 2736 bp. The electrophoresis results showed that about 3000 bp bands were obtained by using Anglu expression cassette as template, the electrophoresis results were consistent with our expectation(Figure 2).
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[[File:Figure 2 Primer design and agarose gel electrophoresis for plasmid identification.png|500px|thumb|center|Figure 2 Primer design and agarose gel electrophoresis for plasmid identification.]]
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<!-- Uncomment this to enable Functional Parameter display  
 
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Revision as of 07:06, 22 October 2020


AnGlu Starch degradation

EXP, AnGlu, XPR2t part will be digested by BsaI and then assembled as the module 1 through the T4 ligase. The module 1 and Leu selection marker will be digested by SapI and then assembled as “EXP-AnGlu-XPR2t-Leu” expression cassette through the T4 ligase. The cassette can be transformed into the Yarrowia lipolytica plotoplast and integrated into the genome for AnGlu efficient expression.



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3683
    Illegal EcoRI site found at 3733
    Illegal EcoRI site found at 4695
    Illegal SpeI site found at 756
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3683
    Illegal EcoRI site found at 3733
    Illegal EcoRI site found at 4695
    Illegal NheI site found at 817
    Illegal SpeI site found at 756
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3683
    Illegal EcoRI site found at 3733
    Illegal EcoRI site found at 4695
    Illegal BglII site found at 2537
    Illegal BglII site found at 5964
    Illegal BamHI site found at 3157
    Illegal XhoI site found at 4678
    Illegal XhoI site found at 4707
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3683
    Illegal EcoRI site found at 3733
    Illegal EcoRI site found at 4695
    Illegal SpeI site found at 756
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3683
    Illegal EcoRI site found at 3733
    Illegal EcoRI site found at 4695
    Illegal SpeI site found at 756
    Illegal NgoMIV site found at 3821
    Illegal AgeI site found at 3364
    Illegal AgeI site found at 5292
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Yarrowia lipolytica can use starch as sole carbon sources when the AnGlu is expressed successfully, which means the AnGlu has the ability to degrade the starch efficiently.

1.Plasmid Construction

We constructed the plasmids according to DESIGN page. There are four parts in our plasmid library: a promoter-EXP (BBa_K3578000), a terminator-XPR2t (BBa_K3578002), expression genes glucoamylase (BBa_K3578001), and the Leu selection marker (BBa_K3578004). By BsaI and SapI IIs enzymes digestion and T4 ligase, we successfully constructed the plasmids which separately carried the AnGlu expression cassette with Leu selection marker (Figure 1). Then plasmids were linearizd with EcoR I and transformed into Y. lipolytica plotoplast. The AnGlu expression cassette with Leu selection marker DNA fragment will be randomly integrated into genome loci.

Figure 1 Schematic diagram of plasmid construction process.

By the PCR experiment, we randomly selected 10 strains in which AnGlu and Leu expression cassette have been successfully integrated into the genome. We designed the primers(F/R) to identify the Anglu expression cassette. The primer design was shown in the figure 2, and the predicted PCR result is 2736 bp. The electrophoresis results showed that about 3000 bp bands were obtained by using Anglu expression cassette as template, the electrophoresis results were consistent with our expectation(Figure 2).

Figure 2 Primer design and agarose gel electrophoresis for plasmid identification.