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Team:Nanjing-China/tran
From 2013.igem.org
Revision as of 07:48, 24 September 2013 by GaoJian NJU (Talk | contribs)
- Transporter
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Currently, Pseudomonas sp. strain ADP seems to be the optimal bacteria strain for atrazine degradations, which appears to be the sole nitrogen source for the bacteria. A study has found that a large plasmid carries all the genes and complex pathways related to atrazine degradation in Pseudomonas sp. strain ADP. We analyzed all the genes in it and finally found a multiple transmembrane protein which we called TRM (Fig. 3-3-1). We hypothesized that the function of transmembrane protein was related to atrazine transportation and thus confirmed it with several experiments.
Fig. 3-3-1 TMHMM posterior probabilities for WEBSEQUENCE. TRM has 11 trans-membrane domains. In prokaryotes, a protein like this has been considered as a potential transmembrane protein.
Experiments and Results
To prove that TRM is a transmembrane protein, we constructed a circuit where a constant promoter drives the expression of the TRM which is fused with a green fluorescent protein to localize it. And the result of transmembrane protein verification is shown in Fig. 3-3-2, suggesting that TRM is located on the cell membrane.
Fig. 3-3-2 The circuit we constructed to verify the function of TRM and subcellular localization of TRM-GFP fusion protein. (A) The circuit we constructed to verify the function of TRM. (B) Fluorescent microscopy image of cells expressing TRM-GFP (green) shows that TRM distributed throughout the cell membrane.
To prove that TRM is related to transportation of atrazine, we used HPLC to analyze the concentration of atrazine after incubating engineered bacteria in 500μM atrazine for 24h (Fig. 3-3-3).It is obvious that bacteria with TRM can reduce the concentration of atrazine, suggesting that TRM plays an important role in transportation of atrazine.
Fig. 3-3-3 Atrazine concentration of K-12 with or without TRM. "0h" stands for control group with no atrazine and the bacteria have been incubated for 0 hour. "Wild Type" stands for Escherichia coli K-12 which ispositive control and "Vector" stands for K-12 which contains a plasmid-pGFP which is negative control. "TRM+" is the experimental group where a plasmid-pGFP which carries TRM was transformed into Escherichia coli K-12. And the result shows that K12 is significantly different from TRM+ in the statistical sense and it also indicates that TRM plays an important role in transportation of atrazine.
