Difference between revisions of "Part:BBa K4399009"
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<partinfo>BBa_K4399009 short</partinfo> | <partinfo>BBa_K4399009 short</partinfo> | ||
| − | Induciable expression box of SbMYB75. | + | Induciable expression box of '''SbMYB75''' ('''BBa_K4399003'''). |
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===Results=== | ===Results=== | ||
| − | The DNA elements (golden gate compatible, P<sub>LexA35S</sub>， SbMYB75, T<sub>hsp18.2</sub>) were synthesized by Gensript based on their sequences and sub-cloned into pUC57 (level-0 vectors). | + | The DNA elements (golden gate compatible, '''P<sub>LexA35S</sub>''' ('''BBa_K3900024''')， '''SbMYB75''' ('''BBa_K4399003'''), '''T<sub>hsp18.2</sub>''' ('''BBa_K4399006''')) were synthesized by Gensript based on their sequences and sub-cloned into pUC57 (level-0 vectors). |
The three level-0 vectors were then used to construct Level-1 vector: pEC47742: P<sub>LexA35S</sub>-SbMYB75- T<sub>hsp18.2</sub>( BBa_K4399009), according to the protocol: | The three level-0 vectors were then used to construct Level-1 vector: pEC47742: P<sub>LexA35S</sub>-SbMYB75- T<sub>hsp18.2</sub>( BBa_K4399009), according to the protocol: | ||
{| class="wikitable" style="margin:auto" | {| class="wikitable" style="margin:auto" | ||
| − | |+ PCR reaction system of level-1 vector | + | |+ PCR reaction system of level-1 vector '''BBa_K4399009''' construction |
|- | |- | ||
! !! volume / μL | ! !! volume / μL | ||
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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 then confirmed by PCR and sequencing ( | + | 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 then confirmed by PCR and sequencing ('''Fig 1'''): |
| − | [[File:BBa K4399009-Fig1.png| | + | [[File:BBa K4399009-Fig1.png|500px|thumb|center| '''Fig 1. Nucleic acid gel electrophoresis results of BBa_K4399009'''. |
Line 1, negative control; | Line 1, negative control; | ||
Line 2, positive control; | Line 2, positive control; | ||
| − | Line 3-6, PCR results of 4 single colonies of BBa_K4399009 using | + | Line 3-6, PCR results of 4 single colonies of BBa_K4399009 using '''P<sub>LexA35S</sub>''' ('''BBa_K3900024''') forward primer and '''SbMYB75''' ('''BBa_K4399003''') reverse primer; |
Line 7, marker. | Line 7, marker. | ||
]] | ]] | ||
Latest revision as of 02:36, 10 October 2022
PLexA35S-SbMYB75-Thsp18.2
Induciable expression box of SbMYB75 (BBa_K4399003).
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 17
Illegal PstI site found at 1437 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 17
Illegal PstI site found at 1437 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1371
Illegal BamHI site found at 805
Illegal BamHI site found at 1107 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 17
Illegal PstI site found at 1437 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 17
Illegal PstI site found at 1437 - 1000COMPATIBLE WITH RFC[1000]
Results
The DNA elements (golden gate compatible, PLexA35S (BBa_K3900024)， SbMYB75 (BBa_K4399003), Thsp18.2 (BBa_K4399006)) were synthesized by Gensript based on their sequences and sub-cloned into pUC57 (level-0 vectors). The three level-0 vectors were then used to construct Level-1 vector: pEC47742: PLexA35S-SbMYB75- Thsp18.2( BBa_K4399009), 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 ul of the liagations were used to transform E. coli. Positive clines were then confirmed by PCR and sequencing (Fig 1):
