Difference between revisions of "Part:BBa K3790160"
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===Introduction===
 
===Introduction===
 
[[File:T--Fudan--ccic8-transparent-logo.png|100px|right|2021 Fudan]]
 
[[File:T--Fudan--ccic8-transparent-logo.png|100px|right|2021 Fudan]]
  
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The fusion protein obtained by linking the N-terminus of Bst to the C-terminal of Sso10b via a (G2S)3 linker.
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===Usage and Biology===
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===Experimental Results===
 
===Experimental Results===
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The length of Sso10b-(G2S)3-Bst DNA was 2085 bp, which is approximately 2100 bp after adding homology arms to both ends for PCR cloning. We isolated the target DNA by gel extraction for subsequent reactions.
  
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The length of xxxx DNA was xxx bp, which is approximately xxx bp after adding homology arms to both ends for PCR cloning. We isolated the DNA of interest by gel extraction for subsequent reactions.
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[[File:T--Fudan--bst-4.jpg|600px|thumb|none|'''Figure 1. PCR assembled Bst fusions.''' The first lane was loaded with DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. The sixth lane (lane-4) was loaded with Sso10b-(G2S)3-Bst DNA.]]
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[[File:T--Fudan--albA1-S.ssb-E.ssb.jpg|600px|thumb|none| '''Figure 2. Assembled DNA binding proteins, albA1, S.ssb, E.ssb.''' The first lane was loaded with DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into cultures and sent for Sanger sequencing. Then, we verified the sequencing results, and used the correct ones for further experiments.]]
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===Reference===
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After ClonExpress homologous recombination reaction, Fast-T1 competent bacteria were transformed with reactants, and spread onto LB plates with antibiotics. We picked colony after 16 h culturing plates at 37℃. We further grow clones in 2 ml LB liquid medium with antibiotics, 37℃ shaking overnight.
  
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Mini-prep was performed the following day, and the resulting plasmids were sent to Sanger sequencing.
  
  
 
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'>'''Sequence and Features'''</span>
 
<partinfo>BBa_K3790160 SequenceAndFeatures</partinfo>
 
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Revision as of 19:23, 21 October 2021

Sso10b-(G2S)3-Bst

Introduction

2021 Fudan

The fusion protein obtained by linking the N-terminus of Bst to the C-terminal of Sso10b via a (G2S)3 linker.


Experimental Results

The length of Sso10b-(G2S)3-Bst DNA was 2085 bp, which is approximately 2100 bp after adding homology arms to both ends for PCR cloning. We isolated the target DNA by gel extraction for subsequent reactions.

Figure 1. PCR assembled Bst fusions. The first lane was loaded with DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. The sixth lane (lane-4) was loaded with Sso10b-(G2S)3-Bst DNA.

After ClonExpress homologous recombination reaction, Fast-T1 competent bacteria were transformed with reactants, and spread onto LB plates with antibiotics. We picked colony after 16 h culturing plates at 37℃. We further grow clones in 2 ml LB liquid medium with antibiotics, 37℃ shaking overnight.

Mini-prep was performed the following day, and the resulting plasmids were sent to Sanger sequencing.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 289
  • 1000
    COMPATIBLE WITH RFC[1000]