Revision as of 17:35, 20 September 2015

FadL-GBP

Introduction

FadL

Fig.1FadL-GBP

The FadL protein (48.8 kDa) is an outer membrane protein of the E. coli, which plays an important role in the uptake of exogenous long-chain fatty acid. The FadL protein has twenty antiparallel β-strands, which form a β-barrel structure and are connected by 9 internal loops and 10 external loops. Due to the β-structure, the FadL protein is able to span the outer membrane multiple times to form a long-chain fatty acid-specific channel. With these properties, the truncated FadL protein can be used as a novel anchoring motif for display of proteins on the E. coli cell surface [1].

GBP

The gold binding polypeptide, abbreviated as GBP, is the three-repeated of following 14 aminoacid sequences: MHGKTQATSGTIQS, which has developed in an E. coli cell-surface display system for several years.[2] The recent computation study demonstrated that multiple repeats of GBP have higher affinity to gold surface because of a high degree of conformational flexibility, which can make the binding much stronger. The mechanism of the connection between GBP and gold metal plane is still unknown. By using Molecular Dynamics (MD), it indicates that GBP, with the structure of antiparallel β-sheet, can recognize gold surface via OH-binding. It seems that the hydroxyl as well as amine ligands on GBP can recognize the atomic lattice of gold, aligning the molecule along the variants of a six-fold axis on the Au (111) surface.[3] A separate study also found out the fact that the water molecules played the important roles for the surface recognition by the side chain and hydration layer of the peptides to reach the better binding [4].

Reference:
[1] Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis, Seung Hwan Lee1, Jong-Il Choi1, Si Jae Park1,†, Sang Yup Lee1,2,* and Byoung Chul Park3(2004)
[2] Molecular characterization of a prokaryotic polypeptide sequence that catalyzes Au crystal formation, John L. Kulp III,a Mehmet Sarikayab and John Spencer Evans, Journal of Materials Chemistry(2004)
[3] Assembly of Gold-Binding Proteins for Biomolecular Recognition, Zareie HM1,2* and Sarikaya M3, Austin Journal of Biosensors & Bioelectronics (2015)
[4] Thermodynamics of Engineered Gold Binding Peptides: Establishing the Structure−Activity Relationships, Urartu Ozgur Safak Seker, Brandon Wilson, John L. Kulp,§ John S. Evans, Candan Tamerler, and Mehmet Sarikaya(2014)

Experiment

Fig.2The DNA sequence length of the FadL-GBP is around 1200～1350 bp. In this PCR experiment, the FadL-GBP products size should be near at 1450～1600 bp.

After receiving the DNA sequences from the gene synthesis company, we recombined each FadL-GBP gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the FadL-GBP. The DNA sequence length of the FadL-GBP is around 1200～1350 bp. In this PCR experiment, the FadL-GBP products size should be near at 1450～1600 bp. The Fig.2 showed the correct size of the FadL-GBP, and proved that we successful ligated the FadL-GBP sequence onto an ideal backbone.

Fig.3FadL-GBP

Application

Fig.4Pcons+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, Pcons + 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.5(a)the results of the test group, which contains the genetic sequence of Pcons+ B0034 + FadL-GBP + B0030 + GFP + Ter

Fig.5(b)displays the consequence of the control group with the genetic sequence, Pcon+ RBS + GFP + Ter

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

Sequence and Features