Part:BBa_K3790160

Sso10b-(G2S)3-Bst

Introduction

The fusion protein obtained by linking the N-terminus of Bst to the C-terminus 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]