Part:BBa_K249019:Experience
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Applications of BBa_K249019
One of the sub-projects for the bioremediation of the tailings ponds is to reduce heavy metals to create magnetic nanoparticles that can then be removed from the pond. To do this we need to be able to produce Mms6 (the iron reducing protein) and show that it can successfully produce the nanoparticles within the Escherichia coli cell. He is the work we have accomplished so far to characterize Mms6.
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Method
In order to characterize the effect of Mms6 production on Escherichia coli DH5α cells, we utilized BioBrick BBa_K249019, a part submitted by the 2009 Lethbridge iGEM team. BBa_K249019 is the Mms6 coding region (BBa_K249016) with a ribosomal binding side (BBa_B0030) and a double terminator (BBa_B0015) under the control of a lactose inducible promoter ( BBa_R0010).
We inoculated two 500 mL cultures of LB media with Escherichia coli DH5α cells containing BBa_K249019 and grew the cells until they reached approximately 0.6 OD600 units. At this point, expression of the Mms6 protein was induced in one of the 500 mL cultures by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). One milliliter of cells from each sample was removed at one hour intervals and the optical density at 600 nm was recorded and plotted.
Results
The addition of IPTG to cells containing the BBa_K249019 BioBrick caused a slowdown of cell growth after 2 hours, and a subsequent plateau at approximately one-half of the cells density that uninduced cells reached after three hours post-induction.
Conclusion
The slowdown in cell growth as a result of production of Mms6 protein could be attributed to a number of factors.
One potential explanation could be a significant diversion of cellular resources to the production of Mms6 protein. This is unlikely, as subsequent analysis of cell fractions with SDS-PAGE showed no increase in banding at the expected molecular weight.
Another explanation could be that the Mms6 protein is toxic to the cells, and causes them to die. This possibility is being explored in collaboration with the Calgary team by using their troubleshooting kit.
References
1Arakaki A., Masuda F., Amemiya Y., Tanaka T., Matsunaga T. (2010). Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria. Journal of Colloid and Interface Science. 343:65-60.