Difference between revisions of "Part:BBa K3790150"

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<partinfo>BBa_K3790150 short</partinfo>
 
<partinfo>BBa_K3790150 short</partinfo>
  
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__TOC__
  
 
===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 C-terminus of Bst Pol to the N-terminal of DbpA via a (G2S)3 linker
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The fusion protein obtained by linking the C-terminus of Bst to the N-terminal of DbpA via a (G2S)3 linker.
  
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__TOC__
 
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===Usage and Biology===
 
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The fusion protein obtained by linking the C-terminus of Bst Pol to the N-terminal of DbpA via a (G2S)3 linker. Moreover, in our experimental expectation, the enzymatic activity of this fusion protein is increased compared to wild-type Bst Pol.
 
  
 
===Experimental Results===
 
===Experimental Results===
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The length of Bst-(G2S)3-DbpA DNA was 2013 bp, which is approximately 2020 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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[[File:T--Fudan--DbpA-bst.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 second lane (lane-1) was loaded with Bst-(G2S)3-DbpA DNA.]]
  
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The length of Bst-(G2S)3-DbpA DNA was 2013 bp, which is approximately 2020 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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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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[[File:T--Fudan--DbpA-bst.jpg|600px|thumb|none| '''Figure 1. Assembled fusion proteins.''' The first lane was loaded with DNA ladder, sizes were marked on the image. The second lane (lane-1) was loaded with Bst-(G2S)3-DbpA DNA. 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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After ClonExpress® homologous recombination reaction, Fast-T1 competent bacteria were transformed with reactants, and spread onto LB plates with ampicillin. We picked colony after 16 h culturing plates at 37℃. We further grow clones in 2 ml LB liquid medium with kanamycin, 37℃ shaking overnight.
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Mini-prep was performed the following day, and the resulting plasmids were sent to Sanger sequencing, our synthesized DNA sequence was confirmed to be correct.
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===Reference===
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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_K3790150 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3790150 SequenceAndFeatures</partinfo>
  

Latest revision as of 17:23, 21 October 2021


Bst-(G2S)3-DbpA

Introduction

2021 Fudan

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


Experimental Results

The length of Bst-(G2S)3-DbpA DNA was 2013 bp, which is approximately 2020 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 second lane (lane-1) was loaded with Bst-(G2S)3-DbpA 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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]