Difference between revisions of "Part:BBa K4399012"
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The reaction ware incubated in a thermocyler for: 37℃ 3 min, 16 ℃ 4 min, 32 cycles; 16℃ 15 min; 50℃ 5 min, 80℃ 5 min. 10 ul of the liagations were used to transform E. coli. Positive clines were confirmed by sequencing and PCR. | The reaction ware incubated in a thermocyler for: 37℃ 3 min, 16 ℃ 4 min, 32 cycles; 16℃ 15 min; 50℃ 5 min, 80℃ 5 min. 10 ul of the liagations were used to transform E. coli. Positive clines were confirmed by sequencing and PCR. | ||
| − | + | [[File:BBa_K4399012-Fig1.png|200px|thumb|center| Figure 1 Protein SDS-PAGE electrophoresis results of OmpR234]] | |
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Revision as of 16:23, 4 October 2022
The inducible anthocyanin biosynthesis system
It is an inducible anthocyanin biosynthesis system, which contains a constitutive GFP expression box, an inducible SbMYB75 expression box, an inducible SbDEL expression box and a constitutive XVE expression box.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Unknown
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal XbaI site found at 696
Illegal XbaI site found at 1579
Illegal XbaI site found at 8415
Illegal PstI site found at 2759
Illegal PstI site found at 4179
Illegal PstI site found at 4264
Illegal PstI site found at 8599
Illegal PstI site found at 8770 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal XbaI site found at 696
Illegal XbaI site found at 1579
Illegal XbaI site found at 8415
Illegal PstI site found at 2759
Illegal PstI site found at 4179
Illegal PstI site found at 4264
Illegal PstI site found at 8599
Illegal PstI site found at 8770 - 1000COMPATIBLE WITH RFC[1000]
Results
Construction of level-0 vectors
The DNA elements (golden gate compatible, promoters: PAtUBI5, PLexA35S, P2×35S; CDS of Genes: GFP, SbMYB75, SbDEL, XVE; Terminators: Tmas, Thsp18.2, Tnos, T35S) were synthesized by Gensript based on their sequences and were sub-cloned into pUC57.
Construction of level-1 vectors
The level-0 vectors were then used to construct Level-1 vectors: pEC47732: PAtUBI5-GFP-Tmas, pEC47742: PLexA35S-SbMYB75- Thsp18.2, pEC47751: PLexA35S-SbDEL-Tnos, pEC47761: P2×35S-XVE-T35S, according to the protocol:
| volume / μL | |
|---|---|
| level-1 empty vector (200 ng/μL) | 1.0 |
| promoter | 1.5 |
| CDS | 1.5 |
| terminator | 1.5 |
| NEB T4 buffer | 1.5 |
| BSA (10×) | 1.5 |
| T4 ligase | 0.5 |
| BsaI | 0.5 |
| ddH2O | 10.0 |
| the whole volume | 20.0 |
The reaction ware incubated in a thermocyler for: 37℃ 3 min, 16 ℃ 4 min， 32 cycles；16℃ 15 min；50℃ 5 min，80℃ 5 min. 10 μl of the liagations were used to transform E. coli. Positive clines were confirmed by sequencing.
Construction of level-2 vectors
Level-2 vectors were constructed based on level-1 vectors:
| IA | NC | |
|---|---|---|
| volume / μL | volume / μL | |
| level-2 empty vector (200 ng/μL) | 1.0 | 1.0 |
| L1-P1 | 1.5 | 1.5 |
| L1-P2 | 1.5 | 1.5 |
| L1-P3 | 1.5 | 1.5 |
| L1-P4 | 1.5 | - |
| ELE-X | 1.5 | 1.5 |
| NEB T4 buffer | 1.5 | 1.5 |
| BSA (10×) | 1.5 | 1.5 |
| T4 ligase (NEB) | 0.5 | 0.5 |
| BsaI | 0.5 | 0.5 |
| ddH2O | 7.5 | 9 |
| the whole volume | 20.0 | 10.0 |
The reaction ware incubated in a thermocyler for: 37℃ 3 min, 16 ℃ 4 min, 32 cycles; 16℃ 15 min; 50℃ 5 min, 80℃ 5 min. 10 ul of the liagations were used to transform E. coli. Positive clines were confirmed by sequencing and PCR.
