Difference between revisions of "Part:BBa K4399009"

 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4399009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4399009 SequenceAndFeatures</partinfo>
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===Results===
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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 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:
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{| class="wikitable" style="margin:auto"
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|+ PCR reaction system of level-1 vector ‘’’pEC47742: P<sub>LexA35S</sub>-SbMYB75- T<sub>hsp18.2</sub>‘’’ construction
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|-
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!  !! volume / μL
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|-
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| level-1 empty vector (200 ng/μL) || 1.0
 +
|-
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| promoter || 1.5
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|-
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| CDS || 1.5
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|-
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| terminator || 1.5
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|-
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| NEB T4 buffer || 1.5
 +
|-
 +
| BSA (10×) || 1.5
 +
|-
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| T4 ligase || 0.5
 +
|-
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| BsaI || 0.5
 +
|-
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| ddH2O || 10.0
 +
|-
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| the whole volume || 20.0
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|}
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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’’’):
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[[File:BBa K4399009-Fig1.png|300px|thumb|center| '''Fig 1. Nucleic acid gel electrophoresis results of BBa_K4399009'''.
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Line 1, negative control;
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Line 2, positive control;
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Line 3-6, PCR results of 4 single colonies of BBa_K4399009 using  PLexA35S forward primer and SbMYB reverse primer;
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Line 7, marker.
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]]
  
  

Revision as of 02:07, 10 October 2022


PLexA35S-SbMYB75-Thsp18.2

Induciable expression box of SbMYB75.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 17
    Illegal PstI site found at 1437
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 17
    Illegal PstI site found at 1437
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1371
    Illegal BamHI site found at 805
    Illegal BamHI site found at 1107
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 17
    Illegal PstI site found at 1437
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 17
    Illegal PstI site found at 1437
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

The DNA elements (golden gate compatible, PLexA35S， SbMYB75, Thsp18.2) 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:

PCR reaction system of level-1 vector ‘’’pEC47742: PLexA35S-SbMYB75- Thsp18.2‘’’ construction
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’’’):

Fig 1. Nucleic acid gel electrophoresis results of BBa_K4399009. Line 1, negative control; Line 2, positive control; Line 3-6, PCR results of 4 single colonies of BBa_K4399009 using PLexA35S forward primer and SbMYB reverse primer; Line 7, marker.