Composite

Part:BBa_K3578011

Designed by: Ruyi Shi   Group: iGEM20_Worldshaper-Nanjing   (2020-09-20)
Revision as of 07:25, 22 October 2020 by Samdong2017 (Talk | contribs)


AnAmyA Starch degradation

EXP, AnAmyA, XPR2t part will be digested by BsaI and then assembled as the module 2 through the T4 ligase. The module 2 and Leu selection marker will be digested by SapI and then assembled as “EXP-AnAmyA -XPR2t-Leu” expression cassette through the T4 ligase. The cassette can be transformed into the Yarrowia lipolytica plotoplast and integrated into the genome for AnAmyA efficient expression.



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3257
    Illegal EcoRI site found at 3307
    Illegal EcoRI site found at 4269
    Illegal SpeI site found at 756
    Illegal PstI site found at 1260
    Illegal PstI site found at 1573
    Illegal PstI site found at 2572
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3257
    Illegal EcoRI site found at 3307
    Illegal EcoRI site found at 4269
    Illegal NheI site found at 817
    Illegal SpeI site found at 756
    Illegal PstI site found at 1260
    Illegal PstI site found at 1573
    Illegal PstI site found at 2572
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3257
    Illegal EcoRI site found at 3307
    Illegal EcoRI site found at 4269
    Illegal BglII site found at 1808
    Illegal BglII site found at 2704
    Illegal BglII site found at 5538
    Illegal BamHI site found at 2731
    Illegal XhoI site found at 2665
    Illegal XhoI site found at 4252
    Illegal XhoI site found at 4281
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3257
    Illegal EcoRI site found at 3307
    Illegal EcoRI site found at 4269
    Illegal SpeI site found at 756
    Illegal PstI site found at 1260
    Illegal PstI site found at 1573
    Illegal PstI site found at 2572
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3257
    Illegal EcoRI site found at 3307
    Illegal EcoRI site found at 4269
    Illegal SpeI site found at 756
    Illegal PstI site found at 1260
    Illegal PstI site found at 1573
    Illegal PstI site found at 2572
    Illegal NgoMIV site found at 3395
    Illegal AgeI site found at 1336
    Illegal AgeI site found at 2938
    Illegal AgeI site found at 4866
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

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

1.The construction and verification of AnAmyA Expression cassette

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-α-amylase (BBa_K3578003), 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 AnAmyA expression cassette with Leu selection marker (Figure 1). Then plasmids were linearizd with EcoR I and transformed into Y. lipolytica plotoplast. The AnAmyA expression cassette with Leu selection marker DNA fragment will be randomly integrated into genome loci.


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