Team:Nanjing-China/parts

This is a summary of all the parts we submitted this year. Details and character can be found on each respective part and
specific testimony can be found in the Result.

1 Introduction： PgolTS-golS-PgolB works as a promoter, which will result in the expression of downstream gene in the presence of gold ions.

A gold-specific sensory protein GolS from Samonella gol regulon was incorporated into E. coli, which in conjunction with an engineered downstream red fluorescence protein allowed the highly sensitive and selective whole-cell detection of gold(III) ions by naked eyes. The putative gold-chaperone, GolB, in the gol regulon was next verified to be specific to gold(I) ions over other metal ions including copper(I). The subsequent display of GolB on E. coli cell surface permitted selective enrichment of gold ions from media containing various thiophilic metal ions. The cell surface-enriched gold(I) was further shown to be easily recovered and the gold-deprived bacteria were capable for re-usage. E. coli bacteria harboring these gold-specific elements from the gol regulon could be a valuable tool for visual detection and facile recycling of gold ions from environmental resources.

GolS is a recently identified gold-specific MerR family transcriptional regulators from Samonella that can effectively differentiate Au(I) from the other two coinage metal ions, Cu(I) and Ag(I). Inspired by the recognition of the gol regulon (Fig. 1a), we tried to develop a gold ion selective induced system in E. coli with the transformed gol cluster and red fluorescence protein to achieve gold ion selective sensing on bacterial cells. As shown in (Fig. 1b), the gene of red fluorescence protein (RFP) was inserted after the golS promoter in the vector pSB1C3, allowing its expression to be driven by GolS, the gold-specific regulator.

The E. coli cells containing the engineering plasmid were cultured in LB medium until OD600=0.6–0.8, then a final concentration of 20 mM Au3+, Cu2+, Cd2+, Zn2+, or Ni2+ was added to the medium. The induced E. coli cells were harvested by centrifugation and re-suspended in PBS buffer (pH 7.4). The results showed that in the presence of gold ions, RFP was significantly expressed and can be visualized by the naked eye (Fig. 1c). Next, the sensitivity of our engineered E. coli cells to gold was investigated. E. coli cells induced by gradient concentrations (0 mM, 0.25 mM, 1 mM, 5 mM, 10 mM and 20 mM) of HAuCl4 (Au3+) were re-suspended in PBS buffer (pH 7.4). The results demonstrated that even the induced concentration of gold ions reached as low as 0.25 mM (Fig. 1d, e), the red fluorescence of E. coli cells was still visible. Different from micromolecule chemosensors design and synthesis by organic chemists, by using this engineered E. coli we achieved highly selective and sensitive recognition of gold ions by a visible and ratiometric fluorescence change on bacterial cells.

Figure1:
a) Genetic organization of the gol locus in the S. typhimurium LT2 genome. b) Genetic organization of the gold inductive RFP expression plasmid in E. coli.
c) A photograph of E. coli cells containing the gold-induced RFP expression plasmid with a concentration of 20 mM each of metal ions, or without metal ions induction and re-suspended in PBS buffer (pH 7.4).
d) Fluorescence measurement of E. coli cells containing the goldinduced RFP expression plasmid after gradient concentrations of HAuCl4 (Au3+) induction and re-suspended in PBS buffer (pH 7.4).
e) A visible photograph of d.

2 Experimental Section Gold selective sensing by engineered E.coli cell
The gold selective sensing plasmid was constructed by two steps and all DNA fragment were amplified from Salmonella typhimurium genomic DNA. First a 200bp DNA fragment including the gene encoding PgolTS was amplified by the primers PgolS1 and PgolS2. The gene encoding golS and PgolB was amplified by the primers GolS1 and PgolB2. Then these two fragments were connected by overlap PCR using PgolS1 and PgolB2. After confirmed by sequencing, the PCR product was digested by EcoRI and XbaI, and then inserted into pSB1C3 vector.

Reference:
[1].Wei Wei, Tianze Zhu, Yue Wang, Hualin Yang, Ziyang Hao, Peng R. Chenand Jing Zhao, Engineering a gold-specific regulon for cell-based visual detection and recovery of gold, Chemical Science, 2012, 3, 1780.