Part:BBa_K3790153

Bst-(G2S)3-albA1

Bst-(G2S)3-albA1

Introduction

The fusion protein obtained by linking the C-terminus of Bst to the N-terminal of albA1 via a (G2S)3 linker.

Experimental Results

The length of Bst-(G2S)3-albA1 DNA was 2082 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 ninth lane (lane-3) was loaded with Bst-(G2S)3-albA1 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]

Experimental Results

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.

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.

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]