Difference between revisions of "Part:BBa K3578011"
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=1.The construction and verification of AnAmyA Expression cassette= | =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. | 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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| + | [[File:Schematic .png|500px|thumb|center|Figure 1 Schematic diagram of plasmid construction process.]] | ||
Revision as of 07:27, 22 October 2020
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.
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.
