Difference between revisions of "Part:BBa K4399009"

 
 
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<partinfo>BBa_K4399009 short</partinfo>
 
<partinfo>BBa_K4399009 short</partinfo>
  
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Induciable expression box of SbMYB75.
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Induciable expression box of '''SbMYB75''' ('''BBa_K4399003''').
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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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>''' ('''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:
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{| class="wikitable" style="margin:auto"
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|+ PCR reaction system of level-1 vector '''BBa_K4399009''' 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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|-
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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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| NEB T4 buffer || 1.5
 +
|-
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| BSA (10×) || 1.5
 +
|-
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| T4 ligase || 0.5
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|-
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| BsaI || 0.5
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|-
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| ddH2O || 10.0
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|-
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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|500px|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  '''P<sub>LexA35S</sub>''' ('''BBa_K3900024''') forward primer and  '''SbMYB75''' ('''BBa_K4399003''') reverse primer;
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Line 7, marker.
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]]
  
  

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:
  • 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 (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:

PCR reaction system of level-1 vector BBa_K4399009 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 (BBa_K3900024) forward primer and SbMYB75 (BBa_K4399003) reverse primer; Line 7, marker.