Part:BBa_K1694037

Pcons+B0034+FadL-GBP+B0030+GFP+J61048

Introduction

Fig.1 P_cons+B0034+FadL-GBP+B0030+GFP+Ter

The transmembrane protein, FadL, fused with the GBP , can act as an anchoring motif for displaying GBP on the surface of E. coli. To achieve the convenient observation, the genetic sequence of green fluorescent protein was introduced in our design. With the dual display of the GBP and the GFP, the E. coli can not only bind on the gold surface but also produce green fluorescent signal.

Experiment

Fig.2 The PCR result of the P_cons+B0034+FadL-GBP+B0030+GFP+J61048. The DNA sequence length is around 2000~2200 bp, so the PCR products should appear at 2200~2400 bp.

After assemble the DNA sequences from the basic parts, we recombined each P_cons+B0034+FadL-GBP+B0030+GFP+J61048 gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the parts. The DNA sequence length of these parts is around 2000~2200 bp. In this PCR experiment, the scFv products size should be near at 2200~2400 bp. The Fig. 2 showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.

Fig.3 P_cons+B0034+FadL-GBP+B0030+GFP+Ter

We intended to make the observation of the result more directly by producing green fluorescence at the same time. To make the comparison of the binding efficiency, we selected the genetic sequence, P_cons + RBS +GFP +Ter, as the negative control group. Because there was no existence of fully-functioned phenomenon, there was no positive control in our experiment.

Fig.4(a) The results of the test group, which contains the genetic sequence of P_cons+ B0034 + FadL-GBP + B0030 + GFP + Ter

Fig.4(b) Displays the consequence of the control group with the genetic sequence, P_cons+ RBS + GFP + Ter

According to the pictures shown above, we can see that there are more E. coli with gold-binding polypeptide being observed on the gold chip than the E. coli without gold-binding polypeptide. It indicates that the E. coli with gold-binding polypeptide can attach on gold chip more effectively than others without gold-binding polypeptide.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 499
  Illegal BamHI site found at 985
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 275
  Illegal NgoMIV site found at 767
  Illegal NgoMIV site found at 1192
  Illegal NgoMIV site found at 2102
  Illegal AgeI site found at 908
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2018