Part:BBa_K3578011
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
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000COMPATIBLE 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.
Results
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.
By the PCR experiment, 10 strains integrating AnAmyA and Leu expression cassette were picked up for the following experiments. We designed the primers(F/R) to identify the AnAmyA expression cassette. The primer design were 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 AnAmyA expression cassette as template, the electrophoresis results were consistent with our expectation(Figure 2).
2.The expression of AnAmyA in Y. lipolytica is sufficient to allow growth on starch
In order to verify whether the AnAmyA possess the ability for starch degradation. Engineered Y. lipolytica expressing the AnAmyA (Polg-AnAmyA) individually were cultured in the starch medium in which the 30 g/L starch was used as the sole carton source. In addition, the strain Po1g was also cultured in the starch medium with the addition of 0.4g/L leucine as the control (Polg-Leu). The OD was measured every 24 hours. As shown in Figure 3, the Polg-AnAmyA almost did not growth in the starch medium. The result can also be directly observed as shown in Figure 4. In summary, Po1g-AnAmyA did not work as expected.
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