Team:Cornell/project/wetlab/mercury

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Wet Lab

Construct Design

To allow for the transport and sequestration of mercury ions into E. coli cells, genes that encode for the cellular production of heavy metal transport proteins and metallothioneins have been added to the pSB1C3 high copy bacterial plasmid. The mercury transport system is composed of merT and merP, genes originally found in Pseudomonas aeruginosa. merP is a periplasmic mercury ion scavenging protein. merT is an integrated membrane protein that works to transport mercury ions into the cell’s cytoplasm.The merT and merP coupled transport system has been used in previous studies to develop luminescence based biosensors for the detection of mercury in the surroundings of bacterial cells.

Our biobrick BBa_K1460004 is composed of the Anderson promoter followed by a ribosomal binding site, merT, merP, and a terminator. The constitutive Anderson promoter allows for the constant expression of metal uptake proteins within our engineered E. coli. The biobrick BBa_K1460007 contains the mercury transport proteins (along with promoter, ribosomal binding site, and terminator) upstream of the GST-YMT metallothionein gene in pSB1C3. By coupling the merT and merP system with metallothionein, we hope to develop an effective biological system for our cells to uptake mercury ions and bind intracellularly to metallothioneins.

BBa_K1460008

Results

Cells successfully expressing merT and merP should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. To test for mercury sensitivity, E.coli BL21 and engineered BL21 with part BBa_K1460004 in the ampr plasmid pUC57 were grown for a 24 hour period in LB with 1 mM, .1 mM and .01 mM Pb. Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (GST-YMT) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the lead sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted by 1/2 with LB + 2 mM Pb for a final lead concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for lead concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.
There was no statistically significant difference between BL21 wild type and BL21 engineered to express merT/merP and GST-YMT in final culture concentration of mercury or mercury sequestered per OD. The mercury concentrations used in this test were much higher than was shown in growth experiments to completely prevent growth of BL21, so it is likely that cells were quickly killed once metal was added, possibly confounding results. To verify this construct is successful in removing mercury from water, we must repeat these experiments using lower concentrations of Hg. We were not able to complete these experiments, however, as the limit of detection of the ICP-AES used to test these metal concentrations is above the

References


  1. Lund, P., & Brown, N. (1987). Role of the merT and merP gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions. Gene, 207-214.
  2. Omura, T., Kiyono, M., & Pan-Hou, H. (2004). Development of a Specific and Sensitive Bacteria Sensor for Detection of Mercury at Picomolar Levels in Environment. Journal of Health Science, 379-383.