Part:BBa_M50437:Experience

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Applications of BBa_M50437

Ideally, this plasmid could be used to speed up synthesis of 2,3-DHB, which would, in turn, solubilize iron.

User Reviews

UNIQ656bdb3ada2c05da-partinfo-00000000-QINU UNIQ656bdb3ada2c05da-partinfo-00000001-QINU

We transformed ordered this construct from DNA 2.0. We used the pD441-CH plasmid backbone, which encodes for kanamycin resistance, and then transformed this plasmid into "E. coli".

Western Blot

We then grew up cells with our plasmid in LB + kanamycin solution with 2 mM IPTG at 37℃ for 2 days, then ran a Western blot assay to see if ahpC and entC were being produced. Our western blot results (figure 1) showed that the entC enzyme was not being produced, or at least not being produced in large enough quantities to effect 2,3-DHB synthesis. Possible explanations for our failure to produce entC include, but are not limited to, a failure in plasmid design or production; a degradation of entC within the cell into smaller segments, explaining the light band on our membrane; or an inability of the cell to carry large amounts of the entC enzyme. No matter the cause, because the two genes work together to produce 2,3-DHB, we concluded that 2,3-DHB was not being produced within our cells, meaning they would not have been able to solubilize a statistically different amount of iron (III) than standard E. coli.

[File:PLTK KEL Western Blot.png]

Iron Toxicity Assay

In order to test iron solubility due to our construct, we decided to grow up our construct in different iron chloride concentrations. We hypothesized that cells creating ahpC and entC without feedback regulation (i.e. with our plasmid) would die due to iron toxicity. In order to test cell viability, we grew up two cultures in LB and kanamycin media at 37℃ for 2 days. For our negative control, the cells were grown up solely in LB and kanamycin. Our experimental cells were grown up in a media that consisted of LB, kanamycin, and 2 mM IPTG in order to fully induce the promoter. After two days, we measured the OD600 of each culture. We tested ten concentrations of FeCl3: 10 mM of FeCl3, 5 mM of FeCl3, 2.5 mM of FeCl3, 1.25 mM of FeCl3, 1 mM of FeCl3, 0.625 mM of FeCl3, 0.5 mM of FeCl3, 0.25 mM of FeCl3, 0.125 mM of FeCl3, 0.0625 mM of FeCl3, and 0 mM of FeCl3 in media. For each concentration of FeCl3, we used two conditions, one where we added cells that had been induced with IPTG and two microliters of IPTG, and one where we added cells that had not been induced with IPTG. All cells were diluted to an OD600 of 0.01 in the well plate. Each test (+/- IPTG with a certain concentration of iron) was performed in triplicate wells, with an additional well for a blank, which contained solely media, IPTG or water, and iron without cells. This plate was then left to grow for two days at 37℃. We then measured the OD600 of the cells after two days to test for growth. A higher OD600 would indicate more growth. The amount of iron remaining in the media was verified with semi-quantitative iron test strips (usage instructions obtained from the manufacturer), which change color in response to Fe2+ and Fe3+ ions, with the resulting color indicative of the approximate concentration range.

Using a t-test comparing our +IPTG results to an expected value of the -IPTG average, we determined that there was only a statistically significant difference at 5 mM. We used two controls: a blank, which controlled for interference in OD600 from media and iron, and a control measurement of cell growth with no IPTG and therefore no construct expression. However, precipitation of iron (III) chloride led to cloudiness of media, interfered with OD600 measurements at concentrations greater than 2.5 mM FeCl3 likely meaning that our statistical significance at 5 mM was simply an experimental error. Overall, our results indicate that there is no significant difference in cell growth for our control and experimental conditions. Therefore, our cells are not suffering from iron toxicity, and are likely not solubilizing iron at high enough levels to die.

Overall, it appears that our construct is not functioning as planned

Stanford Location

Plasmid name: pLysisToKill
DNA2.0 Gene Construct: pD441-CH, customized
Organism: E. coli
Device type: Actuator
Glycerol stock barcode: 0133024475, 0133024500, 0133027012
Box label: BioE44 S18
Physical location: Room 114, Uytengsu Teaching Lab, Shriram Center