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Part:BBa_K1460001:Experience
Designed by: Eric Holmes Group: iGEM14_Cornell (2014-08-14)
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Applications of BBa_K1460001
Cornell 2014 Results
Because successfully expressed metallothionein inhibits growth in microorganisms, we can use growth tests as a tool for determining successful expression of our metallothionein constructs. We transformed BBa_K1460001 into E.coli BL21-AI (which contains an integrated T7 polymerase under control of the araBAD promoter) and grew it and unmodified BL21-AI in LB+.1% L-Arabinose for 24 hours in an incubated plate reader at 37 degrees Celsius. Plotted below is the average OD for three biological triplicates of BL21 and BL21 BBa_K1460001. Plotted OD is corrected for OD of media.
This graph displays statistically significant (student’s two-tailed t-test, p<.05) differences between unengineered BL21 and BL21 engineered to express metallothionein. This data suggests that GST-YMT is being successfully expressed in this engineered strain. Additionally, when working with these cultures for subsequent metal sequestration tests, final culture ODs were consistently observed to be less than that of wild type cells.
When expressed, GST-YMT should confer resistance to heavy metal toxicity. To test whether the construct BBa_K1460001 did in fact confer resistance to engineered cells, we grew engineered and non-engineered cells in different concentrations of mercury that we found allowed normal growth, slightly inhibited growth, and completely inhibited growth in wild type E.coli BL21. These concentrations corresponded to 0.05 uM Hg, 0.5 uM Hg, and 5 uM Hg respectfully. Besides the respective heavy metal concentrations, all media contained LB and .1% L-Arabinose for induction. For convenience, BL21 curves are graphed in pastels and BBa_K1460001 curves are graphed in dark.
When expressed, GST-YMT should confer resistance to heavy metal toxicity. To test whether the construct BBa_K1460001 did in fact confer resistance to engineered cells, we grew engineered and non-engineered cells in different concentrations of mercury that we found allowed normal growth, slightly inhibited growth, and completely inhibited growth in wild type E.coli BL21. These concentrations corresponded to 0.05 uM Hg, 0.5 uM Hg, and 5 uM Hg respectfully. Besides the respective heavy metal concentrations, all media contained LB and .1% L-Arabinose for induction. For convenience, BL21 curves are graphed in pastels and BBa_K1460001 curves are graphed in dark.
For concentrations of Hg that are not completely toxic to cells, we see very similar results as above for growth above in no metal; cells engineered to express metallothionein have growth inhibition when compared to wild type. What is interesting in this experiment, however is the 5 uM concentration of Hg. While we do see that growth is inhibited when compared to the 0.5 uM and 0.05 uM Hg concentrations for the same strain, there is growth. In unengineered BL21 there is none. This suggests that, in fact, our construct is conferring resistance to metal toxicity to engineered cells.
This part was also co-transformed with parts BBa_K1460003, BBa_K1460004, and BBa_K1460005 (all while these parts were in the non-biobrick plasmid pUC57) to create functional matches of the biobrick parts BBa_K1460006, BBa_K1460007, and BBa_K1460008 respectively. Results from these BioBricks can be found on their respective pages.