Difference between revisions of "Part:BBa K3790151"

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===Experimental Results===
 
===Experimental Results===
  
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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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The length of Bst-(G2S)3-S.ssb DNA was 2235 bp, which is approximately 2250 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--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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[[File:T--Fudan--bst-9.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-S.ssb DNA.]]
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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.
  
 
===Reference===
 
===Reference===

Revision as of 17:48, 21 October 2021


Bst-(G2S)3-S.ssb


Introduction

2021 Fudan

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

Usage and Biology

Experimental Results

The length of Bst-(G2S)3-S.ssb DNA was 2235 bp, which is approximately 2250 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-S.ssb 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.

Reference

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]