Difference between revisions of "Part:BBa M50420:Experience"

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Applications of BBa_M50420

This device is intended to be a controllable, cost-effective, and mosquito-specific insecticide. The device produces Cry11Aa when induced by rhamnose and Cry11Aa has been shown to be effective in killing mosquito larvae by targeting their midgut.

User Reviews

UNIQ99e246601a830e5e-partinfo-00000000-QINU UNIQ99e246601a830e5e-partinfo-00000001-QINU

We ordered this part from ATUM and then transformed into E. coli and cultured in LB + kanamycin. Our E. coli were successfully transformed with this plasmid and expressed kanamycin resistance.

Our first experiment was to detect the production of protein, so we followed a Western Blot protocol found in BIOE 44 Course Reader and Lab Manual, using a Bio-Rad Mini-PROTEAN kit to test for the production of Cry11Aa. We utilized mouse antibodies that are specific to the 6xHIS tag to visualize the presence of Cry11Aa protein in our samples. We tested both the supernatant and the lysed cells themselves to test the functionality of our PelB exporter tag.

To set up the experiment, we created liquid cultures of E. coli from our transformed bacteria colony plate. We induced our liquid cultures after 24 hours with a logarithmic scale of concentrations of rhamnose from 0 uM to 1000 uM and included both 2000 uM and 4000 uM to find the dynamic range of the promoter. We used 0 uM rhamnose as our negative control because no protein should be produced with no induction. Induction with 2000 uM rhamnose was our positive control because maximal induction should occur under these conditions according to the plasmid description provided by ATUM.The concentration of cells in each induction was OD600 0.01. After inducing, we waited 24 hours for the cells to produce sufficient quantities of protein before running the Western Blot on an OD600 1.0 of the induced E. coli cells.

We expected the produced protein to be 650 amino acids long and ~73 kDa. As visible from our results, we obtained a distinct band of protein at ~70 kDa in the lane run on lysed cells and 2000 uM rhamnose induction. There are smaller bands of protein in the lanes on either side of the indicated lane, at 1000 uM and 4000 uM rhamnose induction in the lysed cells, which demonstrates that a 2000uM concentration of rhamnose produces maximal protein. From the lack of protein in the lanes from just the supernatant, this test seems to indicate that Cry11Aa was not secreted. It is possible that the cells did not have enough time to produce and export Cry11Aa, so the protein concentration in the supernatant was too dilute to detect.

In order to better quantify the amount of protein produced within the cells, we then performed an Enzyme Linked ImmunoSorbent Assay (ELISA). We performed the ELISA against the 6xHIS tag on the Cry11Aa protein using the protocol and tools from a GenScript His Tag ELISA Detection kit. Transformed E. coli cells were grown and induced with the same procedure outlined above for the Western Blot; however, instead of using a full logarithmic scale, we ran the ELISA on lysed cells that had been induced with 0 uM rhamnose as our background and negative control, and 2000 uM rhamnose, our positive control. To ensure we could get a reading within the dynamic range of the ELISA kit and spectrophotometer, we ran eight samples in the ELISA: dilutions of 100, 102, 103, and 106 with deionized water on two groups of lysed OD600 1.0 transformed E. coli which had been induced at 0 uM and 2000 uM rhamnose respectively. We also ran a set of standards alongside our experiments to ensure the protocol was carried out successfully. Finally, we used a spectrophotometer at a wavelength of 450 nm to quantify the amount of protein in each sample. We also performed an ELISA on lysed OD600 10.0 cells and on supernatant and lysed OD600 1.0 cells that had been induced over a 72 hour period.

After following the protocol for ELISA, we measured color change in the resulting solutions with a spectrophotometer to quantify the amount of protein produced within transformed E. coli. We expected greater protein output levels in the highest rhamnose induction as compared to the negative control. Therefore, we expected to see a lower reading from the spectrophotometer with more concentrated solutions of transformed and induced E. coli. As visible in Figure 5, our data does not suggest any correlation between dilution or rhamnose induction and amount of protein produced, suggesting that a stage of the ELISA protocol interfered with the detection of Cry11Aa. The standards produced expected results, indicating that the protocol was followed properly. It is possible that the protein levels produced by the cells were too dilute to detect alongside background noise from proteins from LB + Kan media.

Therefore, we decided to run the ELISA again with the same protocol, except using ELISA samples with OD600 10.0 of transformed and induced E. coli instead of OD600 1.0. This change in protocol was intended to increase the concentration of proteins and thereby allow for detection by ELISA. As visible in Figure 6, there was still no indication of correlation between induction or dilution and protein production. Therefore, we concluded that protein concentration was not the issue. Another explanation could involve the lysis buffer, which was different than the buffer used in the Western Blot; its interaction with the E. coli cells could potentially have affected Cry11Aa. Another possibility is that the LB + Kan media contained too much protein artifact and skewed the results.

Following the second unproductive ELISA experiment, we decided to run a third one and eliminate the two most likely sources of error, the lysis buffer and the LB + Kan media. We decided to run the ELISA on the supernatant to avoid damaging our protein with lysis, so we gave the cells three days to produce and export protein after inducing them so that sufficient protein would be produced before running the ELISA. We also ran the ELISA on cells of OD600 1.0 that were lysed according to the Western Blot lysis procedure, since this lysis procedure seemed to not affect the protein. We also used EZ + Kan media, which contains no protein artifacts since it is synthetic. We also ran the ELISA on supernatant of 0 uM and 2000 uM rhamnose induced E. coli.

Results from this assay did not indicate a correlation between protein production and induction with rhamnose. As seen in Figure 7, the more diluted the supernatant from induced E. coli had a higher concentration of protein. This is the opposite of what we expected, since dilutions were performed with deionized water, and therefore any His-tagged proteins would become more diluted. The data from lysed cells, visible in Figure 7, also does not show correlation between induction and protein production, similar to the previous two assays. A likely explanation is that there is lots of background signal that obscures the actual signal due to a high level of non-specific interactions. A diluent buffer should most be used to prevent non-specific binding instead of water, which may promote the non-specific binding. Therefore, we cannot conclude anything from this assay that would allow us to quantify protein production.

Stanford Location

Plasmid name: LiT
Vector: pD681-PelB
ATUM Gene #: 328029
Organism: E. coli
Device type: Actuator
Glycerol stock barcode: 0133024435
Box label: BioE44 S18