Difference between revisions of "Part:BBa K4399010"

 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4399010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4399010 SequenceAndFeatures</partinfo>
 +
 +
===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 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:
 +
 +
{| class="wikitable" style="margin:auto"
 +
|+ PCR reaction system of level-1 vector '''BBa_K4399010''' 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'''):
 +
 +
[[File:BBa K4399010-Fig1.png|300px|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 6, positive control;
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Line 7, negative control;
 +
Line 8, marker.
 +
]]
  
  

Revision as of 02:20, 10 October 2022


PLexA35S-SbDEL-Tnos

Induciable expression box of SbDEL.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1266
    Illegal PstI site found at 17
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1266
    Illegal PstI site found at 17
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1266
    Illegal PstI site found at 17
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1266
    Illegal PstI site found at 17
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

The DNA elements (golden gate compatible, PLexA35S， SbDEL, Tnos) 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:

PCR reaction system of level-1 vector BBa_K4399010 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_K4399010. Line 1-5, PCR results of 6 single colonies of BBa_K4399010 using PLexA35S forward primer and SbDEL reverse primer; Line 6, positive control; Line 7, negative control; Line 8, marker.