Difference between revisions of "Part:BBa K4399010"
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<partinfo>BBa_K4399010 short</partinfo> | <partinfo>BBa_K4399010 short</partinfo> | ||
| − | Induciable expression box of SbDEL. | + | Induciable expression box of '''SbDEL'''('''BBa_K4399004'''). |
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===Results=== | ===Results=== | ||
| − | The DNA elements (golden gate compatible, P<sub>LexA35S</sub>， SbDEL, T<sub>nos</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''')， '''SbDEL''' ('''BBa_K4399004'''), '''T<sub>nos</sub>''' ('''BBa_K4399015''')) 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: pEC47751: P<sub>LexA35S</sub>-SbDEL- T<sub>nos</sub>( BBa_K4399010), according to the protocol: | + | The three level-0 vectors were then used to construct Level-1 vector: '''pEC47751: P<sub>LexA35S</sub>-SbDEL-T<sub>nos</sub>''' ( '''BBa_K4399010'''), according to the protocol: |
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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 ('''Fig 1'''): | 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 K4399010-Fig1.png| | + | [[File:BBa K4399010-Fig1.png|500px|thumb|center| '''Fig 1. Nucleic acid gel electrophoresis results of BBa_K4399010'''. |
| − | Line 1-5, PCR results of 6 single colonies of BBa_K4399010 using P<sub>LexA35S</sub> forward primer and SbDEL reverse primer; | + | Line 1-5, PCR results of 6 single colonies of '''BBa_K4399010''' using '''P<sub>LexA35S</sub>''' ('''BBa_K3900024''') forward primer and '''SbDEL''' ('''BBa_K4399004''') reverse primer; |
Line 6, positive control; | Line 6, positive control; | ||
Line 7, negative control; | Line 7, negative control; | ||
Latest revision as of 02:37, 10 October 2022
PLexA35S-SbDEL-Tnos
Induciable expression box of SbDEL(BBa_K4399004).
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1266
Illegal PstI site found at 17 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1266
Illegal PstI site found at 17 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1266
Illegal BglII site found at 678
Illegal BamHI site found at 921
Illegal BamHI site found at 990 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1266
Illegal PstI site found at 17 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1266
Illegal PstI site found at 17 - 1000COMPATIBLE WITH RFC[1000]
Results
The DNA elements (golden gate compatible, PLexA35S (BBa_K3900024)， SbDEL (BBa_K4399004), Tnos (BBa_K4399015)) 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: pEC47751: PLexA35S-SbDEL-Tnos ( BBa_K4399010), 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):
