Difference between revisions of "Part:BBa M50069:Experience"

Methods

DNA Device

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: 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.

Plasmid Transformation into E.coli

The devices were transformed into E. coli using the transformation protocol described in BioE 44 Practical 3, and the E. coli was plated on solid media containing kanamycin to select for successfully transformed cells. Sample Preparation for Experimentation Single colonies were selected with a P200 pipet tip and inoculated in approximately 5mL of liquid LB media containing Kanamycin in plastic 15mL round bottom tubes then placed in an incubator shaker kept at 37 degrees Celsius for overnight growth, this procedure was repeated in advance of every subsequent experiment to ensure cells were fresh and healthy. The original plates were kept in a 4 degree Celsius room to slow growth. Following overnight incubation the liquid cultures were diluted to an OD600 of 0.01 and induced with various concentrations of IPTG (0mM, 0.05mM, 0.125mM, 0.25mM, 0.5mM, 1mM - different combinations of induction concentrations were utilized for different experiments) and allowed to incubate overnight. Approximately 24 hours later OD600 density measurements were taken and then for all experiments the equivalent of 1mL of liquid culture at OD600 of 1 was taken. These standardized cultures were then used for experiments.

Western Blot

We ran was a Western Blot experiment to search for the presence of his-tagged proteins (Ara h2). We followed the “WesternBlotforEcoli” and “SDSPAGE_Western_Protocol” procedures found in the “Western blotting” folder in the BioE 44 folder “Protocols and Resources” in the “Labs” file category. The western was run on cell supernatant and cell lysate for samples containing each device.

ELISA

We ran was an ELISA assay to assess the binding capability of our device-derived Ara h2 protein to wells coated with Ara h2 antibody. The ELISA kit we used was purchased from Elution Technologies, and we followed the procedure that was provided with the kit. We only ran the ELISA on cell lysate samples for one of our devices (simple) after our Western results suggested that the giii secretion sequence could potentially be negatively influencing Ara h2 production. The cells in our samples for the ELISA were lysed with Cell Lytic B sourced from Sigma Aldrich using the procedure detailed on the Sigma Aldrich website. To insure cells were optimally prepared for the Elution Technologies ELISA we also performed the cell lysis protocol for liquid samples detailed in the procedure that accompanied the kit.

Results

Western Blot: Determining the presence of Ara h2 in supernatant and cell lysate.

We performed a western blot on a series of samples to confirm the presence of Ara h2. We designed one of our plasmids with a secretion sequence (export) in the hopes to increase secretion of Ara h2 into the supernatant. To determine if this sequence was effective in causing secretion of Ara h2 we ran the western on supernatant from cultures of cells containing each plasmid (export and simple). We also lysed cells containing each plasmid and ran the western on these samples as well to get a picture of total Ara h2 found in the cell. Our hope in running both supernatant and cell lysate samples was to elucidate the efficacy of the secretion sequence giii. Our device is under the control of an IPTG inducible promoter and because we were unsure of the optimum concentration of IPTG to induce cells with we induced samples 24 hours prior to sample-prep with either 0, 0.05, 0.125, 0.25, or 0.5mM IPTG. We utilized the samples induced with 0mM IPTG as our negative control as our device is unable to produce Ara h2 without IPTG to induce the promoter. We did not utilize a positive control for the first experiment we ran, but concluded that if we got unclear results we would investigate the use of a protein of known size with a 6xHis tag as our positive control for a second experiment. We found that Ara h2 was detected at the highest concentrations in simple cell lysate samples induced with between 0.05mM and 0.25mM IPTG. Ara h2 was detected in export cell lysate samples at all IPTG conditions except for 0mM, however not at as high of concentrations as found in the simple cell lysate samples. The supernatant samples revealed little to no protein except for in simple samples induced with either 0.05 or 0.125mM IPTG. Negative controls are working well; no Ara h2 protein was found in the 0mM IPTG induced samples. The raw data suggests that Ara h2 might be naturally secreted as it is found in the supernatant of simple plasmid samples. Furthermore, it could be that case that the giii secretion sequence added to the export plasmid impairs this natural secretion and perhaps even production as Ara h2 is found at reduced concentrations in cell lysate samples and not at all in supernatant samples. It also seems that IPTG induction concentration above 0.25mM may lead to a concentration of Ara h2 that is toxic to E.coli.

ELISA: Determining the functionality (binding) of device-produced Ara h2

We performed an enzyme-linked immunosorbent assay to determine the functionality and concentration of our device-derived Ara h2 in culture. 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.

STANFORD LOCATION Glycerol stock barcode #: 0133026157 Plasmid name: pArah2(i) Box label: BioE44 S17 Organism: E. coli Device type: Actuator DNA2.0 Gene #: custom version of pD444-SR