Difference between revisions of "Part:BBa M50069:Experience"
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Our guiding experimental question was whether or not E. coli cells could be made to produce the peanut protein Ara h2. Our secondary question was whether or not we could cause E. coli cells to secrete the Ara h2 protein. To answer these experimental questions we designed two devices. Both devices had five conserved parts (Figures 1 and 2): An IPTG inducible inducible T5 promoter sourced from the iGEM registry of parts; a strong ribosomal site sourced from the iGEM registry of parts; the Ara h2 gene taken from the Ara h2 protein sequence in the paper Production of peanut antigen in L.lactis, Glenting, et al., reverse translated from protein sequence to a genetic sequence, and codon optimized for the E. coli organism; a 6x histidine tag attached to the end of the Ara h2 sequence for identification with a western blot sourced from DNA 2.0; and a T5 terminator sourced from DNA 2.0. The second device included a giii secretion tag sourced from DNA 2.0 and added directly before the Ara h2 sequence to hopefully aid in Ara h2 secretion. The two devices were synthesized into plasmids containing a kanamycin resistance cassette by DNA 2.0. | Our guiding experimental question was whether or not E. coli cells could be made to produce the peanut protein Ara h2. Our secondary question was whether or not we could cause E. coli cells to secrete the Ara h2 protein. To answer these experimental questions we designed two devices. Both devices had five conserved parts (Figures 1 and 2): An IPTG inducible inducible T5 promoter sourced from the iGEM registry of parts; a strong ribosomal site sourced from the iGEM registry of parts; the Ara h2 gene taken from the Ara h2 protein sequence in the paper Production of peanut antigen in L.lactis, Glenting, et al., reverse translated from protein sequence to a genetic sequence, and codon optimized for the E. coli organism; a 6x histidine tag attached to the end of the Ara h2 sequence for identification with a western blot sourced from DNA 2.0; and a T5 terminator sourced from DNA 2.0. The second device included a giii secretion tag sourced from DNA 2.0 and added directly before the Ara h2 sequence to hopefully aid in Ara h2 secretion. The two devices were synthesized into plasmids containing a kanamycin resistance cassette by DNA 2.0. | ||
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We performed an enzyme-linked immunosorbent assay to determine the functionality and concentration of our device-derived Ara h2 in culture (Figure 4). Because we detected the highest levels of Ara h2 protein in our simple plasmid samples and due to limited number of wells in the ELISA kit we sourced from Elution Technologies, we decided to only analyze this plasmid. We still employed a range of IPTG induction conditions. Our 0mM IPTG sample and the kit-derived 0ng/mL standard acted as our negative controls and the a series of kit-derived standards of known Ara h2 concentration were utilized as the positive controls and reference points. | We performed an enzyme-linked immunosorbent assay to determine the functionality and concentration of our device-derived Ara h2 in culture (Figure 4). Because we detected the highest levels of Ara h2 protein in our simple plasmid samples and due to limited number of wells in the ELISA kit we sourced from Elution Technologies, we decided to only analyze this plasmid. We still employed a range of IPTG induction conditions. Our 0mM IPTG sample and the kit-derived 0ng/mL standard acted as our negative controls and the a series of kit-derived standards of known Ara h2 concentration were utilized as the positive controls and reference points. | ||
Our ELISA results enforced the data obtained from our initial Western. We found protein present and able to bind to anti-Ara h2 coated wells at all IPTG induction conditions except for 0mM, at highest concentration in the 0.125mM IPTG induced sample. Our negative controls were working well; absorbance quantified in these wells was not significantly difference from the absorbance measured in our blank well (background). Our positive controls were also successful as measured absorbance in those samples matched what was detailed in the Elution Technologies protocol. | Our ELISA results enforced the data obtained from our initial Western. We found protein present and able to bind to anti-Ara h2 coated wells at all IPTG induction conditions except for 0mM, at highest concentration in the 0.125mM IPTG induced sample. Our negative controls were working well; absorbance quantified in these wells was not significantly difference from the absorbance measured in our blank well (background). Our positive controls were also successful as measured absorbance in those samples matched what was detailed in the Elution Technologies protocol. | ||
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Revision as of 04:09, 12 June 2017
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