Composite

Part:BBa_K1460008:Experience

Designed by: Eric Holmes   Group: iGEM14_Cornell   (2014-08-26)


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Applications of BBa_K1460008

Cornell 2014 Results


Part BBa_K1460005 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_K1460008. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005 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.
The chart on the top shows the average final concentration of lead in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005. However, when we consider cell density and plot the amount of metal removed per OD (on the bottom chart) there is a statistically significant difference between the two strains. This data suggests that cells engineered with CBP4 and GST-YMT are in fact able to remove lead ions from water. To the best of our knowledge, this is the first successful lead sequestration by an organism genetically engineered to express a transport protein and metallothionein.

Ideally, this experiment would be run with the OD of both strains remaining the same to prevent changes in metabolite concentrations. This is difficult in this experiment as, as we have shown, cells expressing metallothionein have inhibited growth.

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